Novel bacteriocins, transport and vector system and method of use thereof

ABSTRACT

New bacteriocins capable of inhibiting the growth of bacteria are disclosed, along with methods of obtaining secretion of proteins from lactic acid bacteria, and methods for protecting foodstuffs.

[0001] This application claims priority from U.S. provisionalapplication No. 60/026,257, filed Sep. 5, 1996, incorporated herein byreference in full.

FIELD OF INVENTION

[0002] This invention relates to novel polypeptides, bacteriocins,immunity genes obtained from lactic acid bacteria and a method of usethereof.

BACKGROUND

[0003] With the current consumer demand for fresh (i.e., never frozen)foods, it is important that methods be developed for safe storage ofthese products especially for fresh meats which are manufactured locallybut are marketed around the world. The lactic microflora (lactic acidbacteria) of vacuum packaged meats delays spoilage for weeks or months,as opposed to meats packaged under aerobic conditions which develop aputrefactive microflora that causes spoilage within days.

[0004] Vacuum packaged meats have an extended but unpredictable storagelife dependent on the types of Tactics that dominate the microflora.Meat Tactics can cause severe spoilage problems, such as sulphide odorsor greening by some Lactobacillus species and gas or slime production byLeuconostoc species. Other Tactics exert a preservative effect,extending storage life and enhancing meat safety by competitive growth,by producing organic acids, and by producing antagonistic substancesknown as bacteriocins (peptides or proteins that inhibit the growth ofother, usually closely related, bacteria).

[0005] Nisin is a bacteriocin produced by Tactics used for cheesemanufacture, and is the only bacteriocin licensed for use as a foodpreservative. Nisin is unusual because it is active against a wide rangeof gram-positive bacteria, including the spores of Clostridiumbotulinum; unfortunately, its producer strain does not grow inchill-stored meats, and nisin does not function in meat systems.

[0006] Class II bacteriocins are characterized as small, heat stable,hydrophobic peptides with a high isoelectric point. They are produced asprecursors with an N-terminal extension of 18 to 24 amino acids. Thisextension is cleaved at the C-terminus side of two glycine residues togive the mature bacteriocin. Sequence alignment of the N-terminirevealed a remarkable degree of similarity in their hydropathic profiles(Fremaux et al. 1993).

[0007] The nucleotide sequences of the structural genes for severalclass II bacteriocins have been published, including pediocin PA-1/AcH(Bukhtiyarova et al. 1994, Marugg et al. 1992), sakacin A and P (Holcket al. 1989, Tichaczek et al. 1994), lactacin F (Fremaux et al. 1993,Muriana and Klaenhammer 1991), leucocin A (Hastings et al. 1991),lactococcins A, B, and M (Holo et al. 1991; Stoddard et al. 1992; vanBelkum et al. 1991; van Belkum et al. 1992), plantaricin A (Diep et al.1994) and carno-bacteriocins A, BM1, and B2 (Quadri et al. 1994; Woroboet al. 1994). However, the additional genes necessary for bacteriocinproduction have only been determined for the lactococcins and pediocinPA-1/AcH and, in the case of the some of the lactococcins, the gene forimmunity has also been confirmed. The genetic characterization of thelactococcin and pediocin gene clusters indicates that they have similarfeatures. They both have genes for bacteriocin production in an operonstructure, although the structural and immunity genes for thelactococcins can be transcribed independent of the other genes in theoperon. Furthermore, one of the genes in each of the lactococcin andpediocin operons encodes a protein which belongs to the HlyB-family ofATP-binding cassette (ABC) transporters (Higgins 1992). This protein isthought to be involved in the signal sequence-independent secretion ofthe bacteriocins. Recently, genes encoding proteins which resemblemembers of a two-component signal transduction system have beenidentified which are involved in the expression of plantaricin A andsakacin A (Axelsson et al. 1993; Diep et al. 1994).

SUMMARY OF THE INVENTION

[0008] One aspect of the invention is a new bacteriocin, brochocin-C:peptide A (SEQ ID NO:23), peptide B (SEQ ID NO:25) and its correspondingimmunity peptide (SEQ ID NO:27). Another aspect of the invention is apolynucleotide encoding the brochocin-C operon (SEQ ID NO:21), peptide A(SEQ ID NO:22), peptide B (SEQ ID NO:24), or immunity (SEQ ID NO:26).

[0009] Another aspect of the invention is a polynucleotide encoding anew bacteriocin enterocin 900 (SEQ ID NO:28), a polynucleotide encodingthe first enterocin 900 peptide (SEQ ID NO:29), and the enterocin 900peptide (SEQ ID NO:30).

[0010] Another aspect of the invention is a method for inhibitingpathogenic bacteria by providing a bacteriocin selected from the groupconsisting of brochocin-C and enterocin 900, either as a composition orby providing a bacterial source of brochocin-C or enterocin 900. Forexample, one may inhibit spoilage bacteria in foodstuffs, such as meat,inhibit pathogenic bacteria topically on animals, including humans, andinhibit bacteria infection of fermentation reactors.

[0011] Another aspect of the invention is an expression vector forobtaining secretion of proteins from Tactics, comprising a promoterfunctional in the lactic host, a polynucleotide encoding a divergicinsignal peptide (SEQ ID NO:19), and a structural gene. Another aspect ofthe invention is the vector which comprises a plurality of structuralgenes, each operably linked to a polynucleotide encoding a divergicinsignal peptide.

[0012] Another aspect of the invention is a method to attach bacteriocinstructural and immunity genes to a signal peptide or leader peptide geneso that the bacteriocins can be exported from the host cell.

[0013] Another aspect of the invention is a novel food-grade plasmidthat can be used as a plasmid vector for genes including, but notlimited to, bacteriocins, other polypeptides, enzymes or proteins inorganisms for use in food products or as a probiotic.

[0014] Another aspect of the invention is a method to preserve food byadding bacteriocin-producing bacteria.

BRIEF DESCRIPTION OF FIGURES

[0015]FIG. 1. Deferred inhibition tests against C. piscicola LV17C (A)and C. divergens LV13 (B) by divergin A and carnobacteriocin B2. 1.C.piscicola LV17C containing pMG36e; 2.C. piscicola LV17C containingpRW19e; 3. C. piscicola LV17C containing pJKM14.

[0016]FIG. 2. Schematic representation of the 12.3 kb HindIII insert ofpMJ4 and its subclones. Partial restriction maps of some of the insertsare shown. Not all of the HpaII restriction sites on the insert of pMJ6are indicated. The positions and direction of transcription of lcaA.lcaB. lcaC. lcaD. and lcaE on the insert of pMJ6 are shown. Theasterisks on pMJ20 and pMJ26 indicate frameshift mutations of lcaB andlcaE, respectively.

[0017]FIG. 3. Deferred inhibition of leucocin A transformants with C.piscicola LV17C as the indictor strain (A) and lactococcin Atransformants with L. lactis IL1403 as the indicator strain (B). (A) a.L. gelidum UAL1877-22; b. L. lactis IL1403; c. L. gelidum UAL187-13; d.L. lactis IL1403 (pMJ6); e. L. gelidum UAL187013 (pMJ6). APT was used assolid medium. (B) a. L. geliduin UAL187-22) pMB553); b. L. gelidum UAL187-13 (pMB553); c. L. gelidum UAL187-22; d. L. geldum UAL187-13.Glucose-M17 was used as solid medium.

[0018]FIG. 4. Schematic representation of the two-step PCR strategy toreplace the signal peptide of divergicin A with the double-glycine typeleader peptides of leucocin A, lactococcin A or colicin V. In the firstPCR step, the leucocin A (A and B) or lactococcin A (A) gene was used asa template to obtain a megaprimer containing the leucocin A (A), thelactococcin A (A), or the colicin V leader peptide (B). Thesemegaprimers were used to amplify the divergicin structural and immunitygene in a second PCR step. Divergicin A without a leader or signalpeptide was constructed by first amplifying the region upstream of theleucocin A gene (C) and using the resulting PCR product to amplify thediverigicin gene in the second PCR step. Further information is detailedin the text. Abbreviations: L.P.: DNA encoding the double-type glycineleader peptides; BAC: DNA encoding the mature part of leucocin A orlactococcin A; S.P.: DNA encoding the signal peptide of divergicin A;DIV: DNA encoding the mature part of divergicin A; IMM: immunity genefor divergicin A; S: SacI restriction site; H: HindIII restriction site.

[0019]FIG. 5. Antagonistic activity of L. gelidum 187-22 (A), L. lactisIL1403(pMB500) (B), and E. coli MC4100 (pHK22) (C) transformed withpLED6 (a), pLAD6 (b), pCOD1 (c) or pMG36e (d). In panel (B) alsoantagonistic activity of L. lactis IL1403 transformed with pLAD6 (e). C.divergens UAL278 was used as indicator strain.

[0020]FIG. 6. Detection of antagonistic activity by diverglcin A fusedto the lactococcin A leader peptide in a tricine-SDS-polyacrylamide gel.C. divergens UAL278 was used as the indicator strain by the overlaytest. Lane 1: supernatant of L. gelidum 187-22 carrying pLED1. Lanes 2,3and 4: lysates of E.coli BL21(DE3) containing plasmids pHK22 and pTLA1,pTLA1, pTLA1, or pT713 and pHK22, respectively. Abbreviations: M: maturedivergicin A; P: divergicin A precursor containing the lactococcin Aleader peptide.

[0021]FIG. 7. Colicin V production in L. lactis. Deferred inhibitiontest by L. lactis IL1403(pMB500) transformed with (a) pLEC1 or (b)pMG36e using E.coli DH5α as the indicator strain.

[0022]FIG. 8. Two restriction site maps plasmid pCD3.4. The location ofthe Divergicin structural and immunity genes are marked in B as dvnA anddviA respectively.

[0023]FIG. 9. Bacteriocin activity and growth of Lactobacillus sake 1218in mixed culture with variants of Leuconostoc gelidum at 25° C. in mAPTwith 0.1% glucose and the initial pH adjusted to 5.6. (a) Bacteriocinactivity in arbitrary units (AU) per milliliter of supernatant for mixedcultures of L. sake 1218 and L. gelidum UAL187 (Δ) and mixed cultures ofL. sake 1218 and L. gelidum UAL187-22 (). (b) Growth of L. sake 1218with L. gelidum UAL187(Δ), L. gelidum UAL187-22(∘), and Leuc. gelidumUAL187-13(□).

[0024]FIG. 10. Bacteriocin activity and growth of Lactobacillus sake1218 in mixed culture with variants of Leuconostoc gelidum at 2° C. inmAPT with 0.1% glucose and the initial pH adjusted to 5.6. See FIG. 9for definitions of symbols.

[0025]FIG. 11. Log₁₀ CFU of variants of L. gelidum grown in mixedculture with L. sake 1218 per square centimeter of vacuum-packaged beefstored at 2° C. (Δ), L. gelidum UAL187; (□), UAL-187-13; (∘), UAL187-22.The data represent the means of three trials.

[0026]FIG. 12. Log₁₀ CFU of L. sake 1218 showing growth and survival inmixed culture with variants of L. gelidum per square centimeter ofvacuum-packaged beef stored at 2° C. (), L. sake 1218 alone; (Δ), L.sake with L. gelidum UAL187; (□), L. sake with UAL187-13; (∘), L. sakewith UAL187-22. The solid arrow indicates the sampling time at which asulfide odor was first detected in samples inoculated with L. sake 1218;the open arrow indicates the sampling time at which a sulfide odor wasfirst detected in samples inoculated with L. sake 1218 and L. gelidumUAL187-13 or UAL187-22. The data represent the means of three trials.

[0027]FIG. 13. The method of use of this invention is illustrated by thefollowing schematics. The signal peptide gene or leader peptide gene(s)is illustrated as vertical or horizontal hatching. As schematic Aindicates, a signal or leader peptide is attached to a bacteriocin genedevoid of its natural leader peptide or signal peptide gene. A plasmidcan contain a single bacteriocin with its immunity gene. The spacingbetween the structural gene and the immunity gene is not important andthe immunity gene does not necessarily have to follow the structuralgene providing the immunity gene is also expressed and pr/events thebacteriocin from killing its host. As schematic B illustrates a plasmidcan contain more than one copy of a bacteriocin or more than one type ofbacteriocin. The vector can contain many bacteriocins. In scheme B, theleader or signal peptide genes can be different or the same providingthat leader peptide or signal peptide is compatible with the transportsystem in the cell. If the transport system is not compatible with theleader then a transport system can also be introduced into the vector orplasmid (C) or (D). For multiple bacteriocins or proteins eachstructural gene needs to be attached to a leader or signal peptide.

[0028]FIG. 14. Examples of other leader or signal peptides that could beused in this invention and names of other bacteriocins that couldutilize these signal peptides or other signal or leader peptidesincluded herein. The best host for a vector containing a bacteriocingene with a leader or signal peptide gene attached is the organism fromwhich the leader peptide was derived but other closely related organismsfrequently also work with particular leader peptides. Additionalinformation about these bacteriocins and leader peptides can be obtainedfrom Quadri and associates (1994) or references therein. Comparisons ofthe sequence similarities is also provides. The vertical arrow indicatesthe cleavage site in the prebacteriocins.

[0029] Abbreviations

[0030] The abbreviations in the nucleotide sequences are cytidine (c);adenosine (a); thymidine (t); guanosine (g); and in amino acid sequencesalanine (A); arginine (R); asparagine (N); aspartic acid (D); cysteine(C); glutamine (Q); glutamic acid (E); glycine (G); histidine (H);isoleucine (I); leucine (L); lysine (K); methionine (M); phenylalanine(F); proline (P); serine (S); threonine (T); tryptophan (W); tyrosine(Y) and valine (V).

[0031] Other abbreviations used include: carnobacteriocin 26 (cbn 26);carnobacteriocin A (cbnA); carnobacteriocin B (cbnB); Leucocin A (LeuA); Brochocin-C (Broch C)

DETAILED DESCRIPTION

[0032] Definitions

[0033] The term “gene” used herein refers to a DNA sequence includingbut not limited to a DNA sequence that can be transcribed into mRNAwhich can be translated into polypeptide chains, transcribed into rRNAor tRNA or serve as recognition sites for enzymes and other proteinsinvolved in DNA replication, transcription and regulation. These genesinclude, but are not limited to, structural genes, immunity genes andsecretory (transport) genes.

[0034] The term “vector” used herein refers to any DNA material capableof transferring genetic material into a host organism. The vector may belinear or circular in topology and includes but is not limited toplasmids, food grade plasmids, DNA bacteriophages or DNA viruses. Thevector may include amplification genes, enhancers or selection markersand may or may not be integrated into the genome of the host organism.The term “secretion vector” refers to a vector designed to providesecretion of a protein from the host organism.

[0035] The term “plasmid vector” herein refers to a vector that has beengenetically modified to insert one or more genes.

[0036] The term “signal peptide” herein refers to a N-terminal aminoacid sequence which, when attached to a target polypeptide, permits theexport of the target polypeptide from the cell and cleavage of thesignal peptide. The signal peptide accesses the general proteinsecretion pathway. An example of a signal peptide is the Divergicin Asignal peptide described in amino acid SEQ ID NO:7. Other signalpeptides can be used and are known to those skilled in the are. See SEQID NO:9, SEQ ID NO:11, and SEQ ID NO:13.

[0037] The term “leader peptide” herein refers to a N-terminal aminoacid sequence which, when attached to a target polypeptide, permits theexport of the target polypeptide from the cell and cleavage of theleader peptide. The leader peptides include but are not limited to asequence of 15-24 amino acids that are able to be direct export ofpolypeptides from the cell using the cell's dedicated transport system.The leader peptide sequences shares similarity on their primarystructure and contain a conserved processing site of glycine-glycineresidues or glycine-alanine residues at positions −2 and −1 of theprocessing site. The dedicated transport system includes but is notlimited to the ATP binding cassette (ABC) transporter required forleader peptide-dependent transport. There are many different leaderpeptides that could be used including, but not limited to, leucocin A,Colicin V, carnobacteriocin A, carnobacteriocin B2, enterocin 900 orcarnobacteriocin BM1.

[0038] A “processing peptide” includes both leader peptides and signalpeptides, and may refer to both simultaneously, as used herein.

[0039] The term “cassette” herein refers to a DNA sequence containing aseries of bacteriocin genes and if necessary their respective immunitygenes, appropriate promoters, ribosomal binding site (RBS) andterminating sequences and if necessary other regulatory DNA sequences.The cassette consists of two or more nucleotide sequences encoding astructural (bacteriocin or other substrate) gene linked directly to anN-terminal signal peptide DNA sequence compatible for export through thecell's general export pathway or linked to the leader peptide DNAsequence compatible for export through the dedicated transport system ofthe cell or through a compatible dedicated transport system alsoinserted into a vector used to transform the cell.

[0040] The term “food-arade” herein refers to the origin of the DNAmaterial. Food-grade indicates that a regulatory agency would considerthe substance as coming from a “food” source and therefore suitable forinclusion in food or food products. Organisms that are food-grade, suchas lactic acid bacteria and other established genera of starterorganisms, can be added directly to food without concern forpathogenicity.

[0041] The term “bacteriocin” herein refers to polypeptides and proteinsthat inhibit one or more bacterial species. This includes, but is notlimited to, polypeptides or proteins that were derived from specificstrains of bacteria, proteins that were derived from other types oforganisms or proteins developed through genetic engineering. Thebacteriocin can be bacteriostatic or bactericidal.

[0042] The term “class II bacteriocin” herein refers to a bacteriocinwhich includes but is not limited to small or moderate sizedpolypeptides. This includes but is not limited to heat resistantpolypeptides and heat sensitive polypeptides that do not undergopost-translational modification except for cleavage of the leader orsignal peptide and in some cases formation of disulfide bridges. Thisprotein must have suitable size and properties so that it can beexported from a cell. Class II bacteriocins include, without limitation,carnobacteriocin UAL26, leucocin A, brochocin-C, enterocin 900,divergicin A, carnobacteriocins A and B2.

[0043] The term “class II protein” herein refers to a small protein orpolypeptide which does not undergo post-translational modificationexcept for cleavage of the leader or signal peptide and in some casesthe formation of disulfide bridges. This protein must be a suitable sizeand physico-chemical properties so that it can be exported from a cell.Many such proteins or polypeptides are known. One of ordinary skill inthe art can determine which proteins would be suitable without undueexperimentation. These proteins include, but are not limited to,enzymes, inhibitors that are polypeptides or other regulatorypolypeptides or proteins.

[0044] The term “immunity gene” herein refers to a gene that produces aprotein that protects the host organism against the bacteriocin that itproduces.

[0045] The term “host organism” herein refers to a living bacterium ormicroorganism capable of taking up the plasmid vector, expressing thegenes and producing the desired peptide(s). If the secretion of thedesired polypeptide is required, the host organism must have functionaltransport proteins compatible with the signal or leader peptide attachedto the polypeptide to be exported or it must be able to incorporate thededicated transport protein(s) necessary for the leaderpeptide-dependent export of the substrate generated from vector DNA.Host organism capable of utilizing the divergicin A signal peptide usethe general secretory (sec-) pathway of the cell (for additionalinformation see Pugsley (1993) and Simonen and Palva (1993) andreferences therein).

[0046] The term “transport proteins” herein refers to proteins that arein most cases are incorporated into the cell membrane of the hostorganism and facilitate the export of protein(s) with a signal or leaderpeptide specific for the transport protein to the outside of theorganism. Additional regulatory components, binding sites or enzymes mayalso be required for the functioning of the transporter. The ABCtransporter of a specific protease can cleave the signal or leaderpeptide.

[0047] The term “homologous transporter system” indicates that thetransport system and the leader peptide or signal peptide used to exportpolypeptides arise from the same host.

[0048] The term “heterologous transporter system” indicates that thetransport system and the leader peptide or signal peptide used to exportpolypeptides arise from the different hosts. Divergicin A, for exampleof a signal peptide that can be used in heterologous transport systems.Homologous transporter systems can used in homologous or heterologousbacteria if the transport system is introduced into the host organism.

[0049] The term “meat” herein refers to muscle and fat tissue obtainedfrom animal, fish, fowl or seafood including, without limitation,poultry, cattle, swine, sheep, deer, moose, fish and shellfish. The meatcan be accompanied by bones, skin or internal organs. Meat can includeother additives including but not limited to fillers, dyes,preservatives, natural or artificial flavoring. Meat can be raw, cooked,frozen, cured or canned. The meat would normally but not necessarily bepackaged under vacuum or in a modified atmosphere containing elevatedlevels of carbon dioxide, i.e. vacuum or modified atmosphere (MAP).

[0050] The term “susceptible bacteria” refers to a species or strain ofbacteria that is inhibited by the presence of one or more bacteriocinsin its environment. Preferred susceptible bacteria are inhibited bybrochin-C and/or enterocin 900.

[0051] The term “antibody” refers to antisera, monoclonal antibodies,antibody fragments, single chain antibodies and other functionalequivalents capable of binding a bacteriocin of the invention. Preferredantibodies of the invention are capable of binding specifically to abacteriocin of the invention without significant cross-reactivity withother bacteriocins. Antibodies of the invention are prepared byconjugating the polypeptide to a suitable carrier, such as keyholelimpet hemocyanin, and immunizing a suitable mammal (for example, mouse,rat, horse, goat, rabbit, and the like). It is preferred to employ anadjuvant to obtain an enhanced immune response. After time is permittedfor antibodies to develop, they may be fractionated from blood. Ifdesired, monoclonal antibodies may be prepared by generating hybridomasfrom splenocytes obtained from the immunized animal. Similarly, one maysequence antibodies and determine the sequence of the specific bindingdomain, for preparation of single-chain antibodies and the like.

[0052] The term “mutein” as used herein refers to a conservativevariation of a bacteriocin of the invention. In general, a mutein willhave an amino acid sequence that differs from the native sequence by 1-4amino acid residues (including insertions and deletions). Muteins areeasily prepared using modern cloning techniques, or may be synthesizedby solid state methods. All muteins must exhibit bacteriocinogenicactivity of at least a substantial fraction of the native sequencebacteriocin's activity (although not necessarily against the samesusceptible bacteria), and may be tested using the methods describedbelow.

[0053] General Methods

[0054] We have studied the fundamental characteristics and genetics ofbacteriocin production and applied aspects of bacteriocins in meats. Wehave studied eight new bacteriocins from meat Tactics which showpromising antagonistic activity. We have also developed “bacteriocincassettes” (a series of DNA fragments encoding two or more bacteriocins)that would be equivalent to or better than nisin. The ability to do thisis limited by fragment size at present due to difficulties of cloninglarge fragments of DNA.

[0055] By using the tools and techniques described herein, we havedeveloped a system whereby one can select a range of bacteriocinsagainst target bacteria, using the producer bacterium to deliver theantagonistic effect. This is applicable anywhere that lactic acidbacteria can grow without harming the environment to which they areadded.

[0056] An important area of application for this innovative technique isin the preservation of meats and meat products. This advance will allowproduction of vacuum packaged meats and meat products with a predictableand longer storage life.

[0057] The carnobacteriocins disclosed herein are genetically complexand involve as much as 10 kb of DNA for their production. In contrast,leucocin A, produced by Leuconostoc gelidum, involves 4.5 kb of DNA.Leucocin-producing L. gelidum stops the spoilage of meat bysulfide-producing Lactobacillus sake; it inhibits the growth ofpathogenic Listeria monocytogenes; and, when added to commerciallyproduced ground beef, extends the color and odor storage life of retailground beef.

[0058] Bacteriocins are synthesized in the cells as prepeptidesconsisting of a leader component of 15 to 24 amino acids that is cleavedto release the mature bacteriocin. In addition to this structuralprotein, bacteriocins like leucocin A require an immunity protein forprotection of the cell from its own bacteriocin and two dedicatedsecretion proteins for export of the bacteriocin from the cell.

[0059] Most bacteriocins have dedicated bacteriocin secretion systemsand if their genes are incorporated into another host organism theyusually can not secrete the polypeptide or can only secrete thepolypeptides to a lesser extent. Using the methods described herein anexpanded antibacterial spectrum can be achieved by producing multiplebacteriocins in one bacterium such that the bacteriocins can besecreted.

[0060] We have also identified an important bacteriocin, divergicin A,produced by the meat lactic Carnobacterium divergens. The production ofdivergicin involves only 0.5 kb of DNA, because the leader peptide ofdivergicin accesses the general pathway for protein export from thecell. By fusing the structural and immunity genes of other bacteriocinsbehind the signal peptide of gene sequence of divergicin A, we haveachieved production of bacteriocin(s) by host and heterologous bacteria.Utilizing the cell's secretory mechanism means that the dedicatedsecretory proteins of other bacteriocins do not need to be included inthe bacteriocin cassette and leucocin A and other bacteriocins can beproduced with only 0.5 kb of DNA each instead of 4.5 kb of DNA. This isan important breakthrough for the success of the bacteriocin cassettestrategy.

[0061] We have also been able to produce and export a variety ofbacteriocins or other proteins by placing their respective genesequence(s) behind the divergicin signal peptide sequence in a plasmidand inside meat lactic organisms. This protocol has been tested anddemonstrated to work using Divergicin A signal peptide as a leader toseveral polypeptides including but not limited to Carnobacteriocin B2,collcin V, Leucocin A, Brochocin-C and alkaline phosphatase.

[0062] Carnobacteriocin B2 is a well characterized class II bacteriocinproduced by a 61-kb plasmid from Carnobacterium piscicola LV17. Exportof this bacteriocin depends on a specific ABC (ATP-binding cassette)secretion protein. Divergicin A is a strongly hydrophobic, narrowspectrum bacteriocin produced by a 3.4-kb plasmid from C. divergens LV13with a signal peptide that utilizes the general secretory pathway forexport (Worobo et al., 1995). Fusion of the carnobacteriocin B2structural gene (devoid of its natural leader peptide) behind the signalpeptide of divergicin A permitted production and export of activecarnobacteriocin B2 in the absence of its specific secretion genes. Theimmunity gene for carnobacteriocin B2 was included immediatelydownstream of the structural gene. Correct processing of theprebacteriocin occurred following the Ala-Ser-Ala cleavage site of thesignal peptide. Carnobacteriocin B2 was produced by the wild type strainof C. divergens LV13 and in C. piscicola LV17C, the nonbacteriocinogenicplasmidless variant of the original carnobacteriocin B2 producer strainand other heterologous hosts. Both of the host strains are sensitive tocarnobacteriocin B2 and they both acquired immunity when they weretransformed with this construct.

[0063] An alternative approach to the use of signal peptide Divergicin Awas also tested. Many nonlantibiotic bacteriocins of lactic acidbacteria are produced as precursors with a N-terminal leader peptidethat share similarities in amino acid sequence and contain a conservedprocessing site of two glycine residues in positions −1 and −2 of thecleavage site. A dedicated ATP-binding cassette (ABC) transporter isresponsible for the proteolytic cleavage of the leader peptides andsubsequent translocation of the bacteriocins across the cytoplasmicmembrane. To investigate the role that these leader peptides play in therecognition of the precursor by the ABC translocators, the leaderpeptides of leucocin A, lactococcin A or colicin V were fused todivergicin A, a bacteriocin from Carnobacterium divergens that issecreted via the cell's general secretion pathway. Production ofdivergicin was monitored when these fusion constructs were introducedinto Leuconostoc gelidum, Lactococcus lactis and Escherichia coli thatcarry the secretion apparatus for leucocin A, lactococcins and colicinV, respectively. The different leader peptides directed the productionof divergicin in the homologous hosts. In some cases production ofdivergicin was also observed when the leader peptides were used inheterologous hosts.

[0064] For ABC transporter-dependent secretion in E. coli, the outermembrane protein TolC was required: this is not found in lactic acidbacteria. Using the leader peptide strategy, colicin V was produced inL. lactis by fusing this bacteriocin behind the leader peptide ofleucocin A. By fusing colicin V, which is normally produced by theGram-negative bacterium E. coli, behind the Leucocin A leader peptideand inserting the plasmid into lactic acid bacteria, we have been ableto get lactic acid bacteria to produce and export active colicin V.Similarly, by fusing other bacteriocins behind the leucocin leader, wehave used the leucocin leader to direct the secretion of otherbacteriocins by the leader's dedicated transport system. This is animportant accomplishment because it enables the use of bacteriocins ofGram-negative origin in Tactics (Gram-positive bacteria) or otherGram-positive organisms. For example, this enables the design ofFood-Grade organisms to target Gram-negative pathogens such asSalmonella and E. coli. or for the design of organisms with specificfairly narrow or broad spectra of antibacterial activity.

[0065] The small amount of genetic material required using either theleader peptide or the signal peptide approach for independentbacteriocin expression permits the addition of multiple bacteriocinsinto the vector.

[0066] Chill stored, vacuum packaged beef inoculated withsulfide-producing Lactobacillus sake strain 1218 developed a distinctsulfurous odor within three weeks of storage at 2° C., at which time thebacteria had reached maximum numbers of 10⁶ CFU cm⁻². Co-inoculation ofthe meat with the wild type, bacteriocinogenic (Bac⁺) strain ofLeuconostoc gelidum UAL187 delayed the spoilage by Lb. sake 1218 for upto 8 weeks of storage. Co-inoculation of meat samples with an isogenic,slow growing Bac⁺ variant UAL187-22 or with the Bac⁻ variant UAL187-13did not delay the onset of spoilage by Lb. sake 1218. The study showedthat spoilage of chill stored, vacuum packaged beef by a susceptibletarget organism could be dramatically delayed by the Bac⁺ wild typestrain of Leuc. gelidum UAL187. Inoculation with Lb. sake 1218 can beused as a model system to determine the efficacy of biopreservation ofvacuum packaged meats (Leisner et al., 1996). Using the methodsdescribed herein, other bacteriocins and a food-grade vector, thebreadth of antibacterial activity can be increased and the temperaturerange of protection broadened for this and other food applications.

[0067] The use of the methods described herein will enable the meatindustry to reliably predict the storage life of vacuum packaged freshmeats.

[0068] This same technology can be applied for preservation of animalfeeds such as silage; as animal and human probiotics; as a control forSalmonella in poultry intestines; and for human therapy againstinfections of mucosal tissue where Tactics are acceptable microflora.

[0069] We have identified bacteriocins with a spectrum of antagonisticactivity against both Gram-negative and Gram-positive organisms.Described herein is a method to prepare and use gene cassettes with abroad spectrum of antagonistic activity. Using methods described hereina plasmid containing a cassette of genes containing two or morebacteriocin genes can be constructed and transformed into a hostorganism, resulting in export of the bacteriocins from the cell. Theleader peptide can be specific for the dedicated secretion system(s) ofthe host organism or a common signal peptide suitable for a broaderspectrum of host organisms (i.e. Divergicin A signal peptide).

[0070] Using these strategies, the antibacterial spectrum of theproducer strain can be tailored to target a range of spoilage orpathogenic bacteria, including E. coli and Salmonella. Producer strainsthat grow in the target environment can be selected and specificbacteria can be targeted. Broad range bacteriocins that have beenidentified and characterized will be used as well as other bacteriocinsthat target specific organisms.

[0071] This invention refers to the tailoring of specific lactic acidbacteria that grow in hospitable environments, including human/food,animal feed, the mouth, the gastrointestinal tract of humans andanimals, and the female genital tract. Using the technology of multiplebacteriocin production and delivery using lactic acid bacteria, a rangeof bacteriocins will be produced by the bacteria in situ. The principleof multiple bacteriocin production is based on using signal sequence ofdivergicin A produced by Carnobacterium divergens LV13 or leaderpeptides from other bacteria and fusing structural components ofbacteriocin genes and their immunity genes behind the signal peptide orleader peptide. The bacteriocins that can be exported include, but arenot limited to, several from lactic acid (or closely related) bacteriaand colicin V from Escherichia coli.

[0072] This invention includes, but is not limited to the following:

[0073] A method to export bacteriocins from cells using Divergicin A asthe signal peptide sequence. This method involves fusing the signalpeptide sequence of divergicin A produced by Carnobacterium divergensLV13 to the structural component of a bacteriocin gene devoid of itsleader peptide followed for most bacteriocins by a region containing itsimmunity gene, inserting this into a vector then transforming a hostorganism. For most bacteriocins, its immunity gene must also be includedin the plasmid or vector but its does not have to be directly attachedto either the structural protein or the signal peptide.

[0074] A plasmid vector consisting of four DNA sequences operably linkedtogether. The first sequence encodes a plasmid replication andmaintenance sequence, the second DNA sequence encodes a signal peptideor leader peptide sequence which is attached directly to a third DNAsequence which encodes the polypeptide sequence of a bacteriocin proteindevoid of its leader sequence, the fourth sequence encodes the immunitygene specific for said bacteriocin protein.

[0075] A method to prepare the plasmid vector described above and insertthe vector into the host organism. The host organism possesses atransport pathway which utilizes the signal peptide encoded by thesignal peptide sequence.

[0076] A plasmid vector, pCD3.4 (SEQ ID NO:14), which is a food-gradeplasmid and method of use thereof.

[0077] A plasmid vector as described above wherein the signal peptidesequence is SEQ ID NO:7.

[0078] A plasmid vector as described above wherein the bacteriocin andimmunity gene are class II bacteriocin.

[0079] A plasmid vector consisting of three DNA sequences operablylinked together. The first sequence encodes a plasmid replication andmaintenance sequence, the second DNA sequence encodes a signal peptideor leader peptide sequence which is attached directly to a third DNAsequence which encodes the polypeptide sequence of a Class Type IIprotein or polypeptide devoid of its leader sequence.

[0080] An insertion vector as described above wherein the third DNAsequence encodes an enzyme.

[0081] A plasmid vector containing at least five DNA sequences operablylinked together. The first sequence encodes a plasmid replication andmaintenance sequence, the second DNA sequence encodes a signal peptidewhich is attached directly to a third DNA sequence which encodes thepolypeptide sequence of a bacteriocin protein, the fourth sequenceencodes the immunity gene specific for said bacteriocin protein and thefifth sequence encodes a polypeptide sequence for a transport proteinsystem compatible with the signal peptide.

[0082] A method as described above wherein the plasmid contains morethan one bacteriocin.

[0083] A plasmid vector as described above wherein the sequence encodingfor the transporter system is the Leucocin A transporter system and theleader is from Leucocin A.

[0084] The signal peptide or leader peptide for the methods describedabove can be selected from leucocin A, lactococcin A, divergicin A,colicin V or other sequences described herein or any other dedicatedsecretion proteins that are compatible with the host organism.

[0085] A novel plasmid pCD3.4 (SEQ ID NO:14) for transforming food gradebacteria.

[0086] A method to preserve beef by adding Leuconostoc gelidum UAL187.

[0087] A method of preserving meat using food grade bacteriumgenetically modified with an plasmid vector containing one or morebacteriocins.

[0088] A method wherein plasmid vector is pCD3.4 (SEQ ID NO:14) is usedas a vector.

[0089] A method for using food grade bacterium for the protection orpreservation of food.

[0090] A method for using food grade bacterium transfected with a vectorcontaining one or more bacterium for the protection or preservation offood.

[0091] A method for treating bacterial infections in animals or humansusing food grade bacterium containing a naturally occurring bacteriocin.

[0092] A method for treating bacteria infections in animals or humansusing food grade bacterium which has been genetically modified asdescribed herein using one or more bacteriocins.

[0093] A method for treating bacteria infections in animals or humansusing a food grade bacterium which has been genetically modified asdescribed herein.

[0094] A method to inhibit the growth of gram-negative and/or grampositive bacteria using one or more bacteriocins.

[0095] A method to inhibit the growth of gram-negative and/orgram-positive bacteria using a genetically modified host organism.

[0096] Brochocin-C bacteriocin genes and methods of use thereof.

[0097] Enterocin 900 bacteriocin genes and methods of use thereof.

[0098] A method to export class II polypeptides using a leader peptidesequence.

[0099] A method to export class II polypeptides using a signal peptidesequence.

[0100] Novel bacteriocins and leader peptides and a method of usethereof.

[0101] Method of using Leucocin A transporter genes.

[0102] A food-grade plasmid and method of use thereof.

[0103] A method to increase the shelf life of meat.

[0104] A method to test organisms for preservation of meat, dairyproducts or other food products.

[0105] A method to purify certain bacteriocins.

[0106] A method to export bacteriocins using a leader peptide sequence.

[0107] A method to export other polypeptides using a leader peptidesequence.

[0108] A method to introduce immunity to particular bacteriocins intohost organisms.

EXAMPLES

[0109] The following examples are provided as a guide for those of skillin the art, and are not to be construed as limiting the claimedinvention in any way.

Example 1 (Bacteriocins, Sources, Methods of Propagation)

[0110] Table 1 describes many different bacterial strains and plasmids,the bacteriocins they contain and references which provide additionalinformation about the bacterocin or bacterial strain. For information onthe best method to grow a particular organism refer to the appropriatereference or reference therein.

Example 2 (Use of Signal Peptide to Direct the Secretion of Substrates)

[0111] Example using Divergicin A signal peptide and Carnobacteriocin B2as substrate:

[0112] Bacterial Strains and Media.

[0113] Bacterial strains and plasmids used in this study are listed inTable 1. Carnobacteria were grown in APT broth (Difco Laboratories,Detroit, Mich.) at 25° C. without agitation. E. coli was grown in LuriaBertani (LB) medium at 37° C. on a rotary shaker. Agar plates were madeby addition of 1.5% (wt/vol) agar to broth media. Antibiotics were addedas selective agents when appropriate, as follows: erythromycin 200 μg/mland ampicillin 100 μg/ml for E. coli and erythromycin 10 μg/ml forcarnobacteria. Stock cultures of the bacterial strains were stored at−70° C. in the appropriate broth containing 20% (vol/vol) glycerol.

[0114] Oligonucleotide Primer Synthesis and Amplification Reactions:

[0115] In the 3′ region of the nucleotide sequence encoding the signalpeptide of divergicin A there is a HindIII restriction site located 10nucleotides upstream of the sequence encoding mature divergicin A(Worobo et al, 1995). A 35-mer oligonucleotide designed to facilitate anin-frame fusion between the signal peptide of divergicin A and thestructural gene of carnobacteriocin B2 was synthesized on a DNAsynthesizer (Applied Biosystems 391 PCR Mate) for use as a PCR primer(JMc7; 5′-CCCAAGCTTCTGCTGTAAATTATGGTAATGGTGTT-3′)(SEQ ID NO:40). Thefirst 9 nucleotides of JMc7 regenerate the HindIII restrictionendonuclease cleavage site followed by nucleotides encoding thecarboxy-terminus of the divergicin A signal peptide. The last 21nucleotides of the primer are complementary to the 5′ sequencecorresponding to the N-terminal sequence of the carnobacteriocin B2structural gene (cbnB2) immediately following the Gly-Gly cleavage siteof the leader peptide. The reverse primer for the PCR amplification(ImmR) was based on the 3′ nucleotide sequence of the immunity gene forcarnobacteriocin B2 (cbiB2) and contains an overhang of 9 nucleotides toaccommodate an XbaI restriction endonuclease site (Pugsley, 1993). DNAwas amplified in a 100 μl reaction using a temperature cycler (OmniGene,InterSciences Inc., Markham, Ont.). PCR mixtures contained 1.0 μM ofeach primer, 200 μM of dNTPs, 5 mM MgCl2, 2.5 units of Tli DNApolymerase (Promega) and 1× reaction buffer (Promega). pLQ24 was used astemplate DNA for the reaction (Pugsley, 1993). DNA was amplified with 36cycles (denaturation, 93° C., 1 min; annealing, 48° C., 1 min;extension, 75° C., 2 min) followed by a final extension step at 75° C.for 5 min.

[0116] DNA Isolation, Manipulation and Sequence Determination:

[0117] Isolation of plasmid DNA from E. coli and carnobacteria was doneusing the methods described by Sambrook et al, 1989, and Worobo et al,1994. Miniprep plasmid extractions for E. coli MH1 included aphenol-chloroform step which was necessary for restriction endonucleaseanalysis. Standard methods were used for restriction enzyme digestion,ligations, gel electrochoresis and E. coli transformation (Sambrook etal, 1989). Transformation of carnobacteria was done as described byWorobo and associates (1995). DNA was sequenced by Taq DyeDeoxy Cyclesequencing (Applied Biosystems 373A). Sequences were determinedbidirectionally in pUC118 using universal primers.

[0118] Production of and Immunity to Divercicin A and CarnobacteriocinB2:

[0119] Carnobacteria transformed with either pRW19e or pJKM14 weretested for bacteriocin production using the deferred antagonism assay asdescribed by Ahn and Stiles (1990) and references therein. Strainscontaining pMG36e were used as negative controls. Immunity to divergicinA and carnobacteriocin B2 was determined with the transformants asindicators in deferred inhibition assays. To confirm that the zones ofinhibition were caused by a proteinaceous compound, they wereinactivated by spotting Pronase E (1 mg ml-1; Sigma) prior tooverlayering with the sensitive indicator strain.

[0120] Purification and N-Terminal Sequencing of Carnobacteriocin B2:

[0121] Partial purification of carnobacteriocin B2 was done with a 1%inoculum of an overnight culture of C. divergens LV13 containing pJKM14grown in 2 liters of APT broth for 21 h maintained at pH 6.2 with a pHstat (Chem-Cadet; Cole Palmer). The culture was heated (70° C., 35 min)and cells were removed by centrifugation. Supernatant was loaded onto anAmberlite XAD-8 column (4×40 cm; BDH Chemicals, Poole, England)equilibrated with 0.05% trifluoro-acetic acid (TFA). The column waswashed successively with 3 liters of 10, 35 and 40% ethanol. C.divergens LV13 containing pJKMl4 produces carnobacteriocin B2 anddivergicin A, hence C. divergens LV13 was used as the sensitiveindicator strain to eliminate inhibition zones produced by divergicin A.The active fraction was eluted with 3 liters of 50% ethanol. Thisfraction was concentrated by rotary evaporation to approximately 50 ml,and 10 ml was applied to a Sephadex G-50 column (2.5×120 cm, Pharmacia)with a running buffer of 0.05% TFA. Contents of tubes with inhibitoryactivity were collected, pooled and concentrated by rotary evaporationto 1 ml. Various amounts of partially purified carnobacteriocin B2 weresubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and blotted onto polyvinylidene difluoride membrane(Bio-Rad). A duplicate polyacrylamide gel was washed twice with 1 literof water and the gel was placed onto an APT plate and overlayered withsoft APT agar inoculated with 1% of C. divergens LV13. The bandcorresponding to inhibitory activity was excised from the membrane andused for N-terminal sequencing by Edman degradation as described byWorobo et al. (Worobo et al, 1995).

[0122] Construction of Plasmids pRW19e and pJKM14.

[0123] The bacteriocino-genic plasmids pRWl9e and pJKMl4 wereconstructed for use in this study. Both plasmids are derivatives of thelactococcal expression vector pMG36e (Van de Guchte et al, 1989) andtranscription of the bacteriocin genes is under control of the P32promoter for construction of pRW19e, a 514-bp EcoRV-AccI fragment ofpCD3.4 (SEQ ID NO:14) containing both the structural and immunity genesfor divergicin A (Worobo et al, 1995) was cloned into the SmaI and AccIsites of pMG36e. When C.piscicola LV17C was transformed with pRW19e theinhibitory spectrum matched that of C. divergens LV13 (Table 5). Zonesof inhibition were inactivated by pronase E. C.piscicola LV17C withpRW19e also acquired immunity to divergicin A (Table 5). Forconstruction of pJKM14, a 528-bp fragment was amplified by PCR frompLQ24 using the primers JMc7 and ImmR. This fragment was cloned intotheHindIII and XbaI sites of pUC118 to create the plasmid pJKMO5 andsequenced in both directions to confirm the fidelity of the reaction. Aninternal EcoR1 site located in the 5′ region of cbiB2 was utilized togenerate two subclones for completion of the overlapping sequence. Noerrors were detected in the nucleotide sequence compared with nucleotidesequence of the structural and immunity genes for carnobacteriocin B2(Quadri et al, 1994). The 528-bp fragment was excised from pJKMOS usingHindIII and KpnI and cloned into these sites in pRW19e, replacing thedivergicin A structural and immunity genes. The SacI-EcoR1 fragment frompJKM14 containing the fusion between the divergicin A signal peptide andthe carnobacteriocin B2 structural gene was cloned into pUC118 andsequenced to confirm that the correct reading frame was maintained.

[0124] Production of and Immunity to Divergicin A and CarnobacteriocinB2.

[0125] Production of divergicin A and carnobacteriocin B2 was detectedby deferred antagonism assay against sensitive indicator strains. C.piscicola LV17C and C. divergens LV13 were transformed with the plasmidspMG36e, pRW19e and pJKM14 to compare differences in bacteriocinproduction with the divergicin A signal peptide. Results of deferredinhibition tests are shown in FIG. 1 and Table 5. C. divergens Lv13 ismore sensitive to carnobacteriocin B2 than C. piscicola LV17C shown bythe large inhibitory zone in FIG. 1B. Zones of inhibition for wild typestrains and strains containing pMG36e were identical. When C. piscicolaLV17C was transformed with pRW19e, divergicin A was produced asindicated by inhibition of strains sensitive to divergicin A. Noactivity was detected against C. divergens LV13. The wild typecarnobacteriocin B2 producer C. piscicola LV17B produces at least twobacteriocins (Quadri et al, 1994) making comparison between theinhibitory spectra of C. piscicola LV17B and C. piscicola LV17Ccontaining pJKM14 difficult to interpret. To confirm the identity of theinhibitory substance produced by C.divergens LV13 containing pJKM14, thebacteriocins were purified and N-terminal amino acid sequence of theprobable carnobacteriocin B2 peak was determined and shown to beVal-Asn-Tyr-Gly-Asn-Gly-Val. This sequence matches the mature sequenceof carnobacteriocin B2 indicating that the inhibitory substance was infact carnobacteriocin B2, and that proper processing of the bacteriocinoccurred following the Ala-Ser-Ala processing site of the divergicin asignal peptide (SEQ ID NO:1). The nucleotide and amino acid sequence ofthe divergicin A signal peptide is shown fused to the structural gene ofcarnobacteriocin B2 devoid of its natural leader peptide (see SEQ IDNO:34 for full details of the carnobacterium B2 genes and sequences).The sequence for the mature carnobacteriocin B2, locations of theforward primer (JMc7) used for PCR and the HindIII restriction site areindicated. Furthermore, production of carnobacteriocin B2 from pJKM14was also accomplished in the two meat isolates C. divergens AJ and C.piscicola UAL26, and in Lactococcus lactis subsp. lactis IL1403. Usingthis strategy production of Leucocin A, Brochocin-C and Colicin V wasachieved.

[0126] There are a large number of plasmids that could be used in placeof the plasmids described herein. One of ordinary skill in the art canidentify other suitable plasmids and insert the various combinations ofother gene sequences described herein into one of these plasmids withoutundue experimentation.

[0127] Using a Signal Peptide Gene to Export Alkaline Phosphatase fromthe Host:

[0128] Using the procedure described herein and the Divergicin A signalpeptide gene attached to alkaline phosphatase structural gene, theinventors were able to export active alkaline phosphatase from the hostorganism, E. coli. For amplification of the DNA encoding the mature partof alkaline phosphatase, primers KLR 179(5′-GCGCAAGCTTCTGCTCGGACACCAGAAATGCCTGTT-3′) (SEQ ID NO:41) and KLR 180(5′-GGCCAAGCTTGCCATTAAGTCTGGTTGCTA-3′) (SEQ ID NO:42) were used with theE. coli C₄F₁ (Torriani, 1968) alkaline phosphatase gene as a template.Cloning of alkaline phosphatase was essentially as described in example2 for Carnobacteriocin B2 and Worobo et al. 1995.

[0129] Assay for Alkaline Phosphatase:

[0130] Cells from 1.5 ml of an overnight culture grown in LB broth werecentrifuged (9000× g, 5 min, 25° C.) and washed in an equal volume ofSTE (50 mM NaCl, 10 imM Tris pH 8.0, 1 mM EDTA pH 8.0). The culturemedia and periplasmic fractions were assayed for alkaline phosphatase.Periplasmic fractions were prepared by resuspending the washed cells in0.5 ml of 20% sucrose with 50 μl of 0.5 M EDTA and 25 μl of lysozyme (10mg/ml) and incubating at room temperature for 15 min. The samples werecentrifuged (9000× g, 5 min, 25° C.) and the supernatant was assayed foralkaline phosphatase activity (Torriani, 1968) by absorbance at 405 nm.

Example 3 Use of Leader Peptides to Direct Secretion of Substrates viaDedicated Transport System

[0131] Bacterial Strains and Media.

[0132]C. divergens LV13 (Worobo et al., 1995), C. divergens UAL278(McCormick et al., unpublished), L. gelidum 187-13 and L. gelidum 187-22(Hastings and Stiles, 1991), and Pediococcus pentosaceus FBB63C (Grahamand McKay, 1985) were grown in APT broth (All Purpose Tween; DifcoLaboratories Inc.) at 25° C. and 30° C., respectively. L. lactis IL1403(Chopin et al., 1984) and L. lactis IL1403(pMB500) (van Belkum et al.,1989) were grown in glucose-M17 broth (Terzaghi and Sandine, 1975) at30° C. E. coli strains MH1 (Casadaban and Cohen, 1980), DH5 (BRL LifeTechnologies Inc.), BL21(DE3) (Studier and Moffat, 1986), MC4100(Casadaban, 1976), and ZK796 (Wandersman and Delepelaire, 1990) weregrown in TY broth at 37° C. (Rottlander and Trautner, 1970). Solidplating media were prepared by adding 1.2% (wt/vol) agar to the brothmedia. C. divergens UAL278 cells propagated on agar medium wereincubated under anaerobic gas mixture of 90% N2 and 10% CO₂ . E. colistrains transformed with the colicin V encoding plasmid pHK22 (Gilson etal., 1987) were grown in media that contained 0.2 mM 2,2′-dipyridyl toincrease expression of the colicin V operons. When appropriate,antibiotics were added to the media at the following finalconcentrations: erythromycin (200 (g/ml), ampicillin (150 (g/ml),tetracycline (15 (g/ml) and chloramphenicol (25 (g/ml) for E. coli;erythromycin (5 (g/ml) for L. lactis, C. divergens and L. gelidum; andkanamycin (50 (g/ml) for L. lactis.

[0133] Bacteriocin Assay.

[0134] Bacteriocin production was tested as described previously (vanBelkum and Stiles, 1995). To detect divergicin A production, a strain ofC. divergens UAL278 that is resistant to leucocin A was used as anindicator. This resistant strain was isolated by exposing it to asublethal concentration of leucocin A. C. divergens LV13, L. lactisIL1403, P. pentosaceus FBB63C and E. coli DH5 were used as indicatorstrains for leucocin A, lactococcin A, pediocin PA-1 and colicin V,respectively. In some cases, bacteriocin activity was also tested byspotting serial dilutions of the growth medium onto an indicator lawn.

[0135] Purification and N-terminal Sequencing of divergicin A.

[0136] To purify divergicin A from transformants of L. gelidum 187-22, a1% inoculum of an overnight culture was grown in APT broth, which wasmaintained at pH 5.5 using a pH stat (Chem-Cadet; Cole Palmer). After 18h, the culture was heated at 70° C. for 35 min and centrifuged for 10min to remove the cells. The supernatant was loaded onto an AmberliteXAD-8 column (4 cm×40 cm; BDH Chemicals) equilibrated with 0.05%trifluoroacetic acid (TFA) The column was washed with equal volumes of0.05% TFA, and 10%, 35%, and 45% ethanol in 0.05% TFA. The activefraction of divergicin was eluted with 50% ethanol in 0.05% TFA andconcentrated 10-fold by rotary evaporation. Samples of 10 ml were loadedonto a Sephadex G-50 column (2.5 cm×120 cm; Pharmacia) that wasequilibrated with 0.05% TFA. The active fraction was applied to aSDS-polyacrylamide (15%) gel for polyacrylamide gel electrophoresis(PAGE). After electrophoresis, the gel was fixed in 50% methanol and 10%acetic acid for 30 min, washed twice for 1 h with 1 liter of deionizedwater and overlayered on an APT plate with soft APT agar (0.7% wt/vol)inoculated with 1% of a C. divergens UAL278 culture to screen fordivergicin activity. Another sample of the partially purified divergicinobtained from the Sephadex G-50 column was subjected to SDS-PAGE andelectroblotted onto a polyvinylidene difluoride membrane (Bio/Rad) andthe protein band corresponding to the inhibitory activity of theoverlayer test was excised from the gel and used for N-terminalsequencing by Edman degradation, as previously described (Worobo et al.,1995).

[0137] Molecular Cloning.

[0138] Cloning and DNA manipulations were performed as described bySambrook et al. (1989). Plasmid DNA from E. coli was isolated asdescribed by Birnboim and Doly (1979). With some modifications (vanBelkum and Stiles, 1995), the same method was used to isolate plasmidDNA from L. gelidum and L. lactis. Restriction endonucleases, Tli DNApolymerase, the Klenow fragment of E. coli DNA polymerase I, and T4 DNAligase were obtained from Promega, Bethesda Research Laboratories,Boehringer GmbH, or New England Biolabs, and used as recommended by thesuppliers. Competent E. coli cells were transformed as described byMandel and Higa (1970). Electrotransformations of L. lactis and L.gelidum were done according to the methods of Holo and Nes (1989) andvan Belkum and Stiles (1995), respectively.

[0139] Construction of Plasmids.

[0140] A two-step PCR strategy (FIG. 4) was used to obtain a fusionbetween the leucocin A leader peptide and divergicin A. DNA encoding theleucocin A leader peptide and a 176-bp upstream region was amplified byPCR using plasmid pMJ3 (van Belkum and Stiles, 1995) as a template andMB32 (5′-AATTCGAGCTCGCCCAAATC-3′) (SEQ ID NO:43) that is complementaryto the upstream region, and MB37(5′-TGAGTAATTTTCGGTGCAGCACCTCCTACGACTTGTTCGA -3T) (SEQ ID NO:44) that iscomplementary to the leucocin A leader and divergicin A sequence, asprimers. This PCR fragment was subsequently used as a megaprimer toamplify the structural gene encoding divergicin A and a downstreamregion that includes the immunity gene for divergicin, with pCD3.4 (SEQID NO:14) (Worobo et al., 1995) as a template and RW58(5′-TACGCGCAAGAACAGACAAAATC-3′) (SEQ ID NO:45) as the reverse primer.Using the SacI restriction site of MB32 and a HindIII restriction site390-bp downstream of the immunity gene the resulting PCR fragment wascloned into plasmid pMG36e (van de Guchte et al., 1989), giving plasmidpLED1. In a similar way, the sequence encoding the lactococcin A leaderpeptide and a 375-bp upstream region was fused to the gene encodingdivergicin A, except that in the first PCR step, plasmid pMB553 (vanBelkum et al., 1991a) was used as a template and MB38(5′-TGAGTAATTTTCGGTGCAGCTCCTCCGTTAGCTTCTGAAA -3′) (SEQ ID NO:46) that iscomplementary to the lactococcin A leader and divergicin A sequence, andMB39 (5′-TACGAATTCGAGCTCGCCC -3′) (SEQ ID NO:47) that is complementaryto the upstream region, were used as primers. The PCR product containingthe resulting gene fusion was cloned into the SacI and HindIII sites ofpMG36e, giving plasmid pLAD6. Plasmid pCODl, that contains a gene fusionbetween the colicin V leader sequence and divergicin A, was constructedin an identical way to pLED1, except that MB42 was used as a PCR primerinstead of MB37. MB42(5′-ATTTTCGGTGCAGCACCTCCAGAAACAGAATCTAATTCATTTAGAGTCAGAGTTCTCATAATAACTTTCCTCTTTT-3′) (SEQ ID NO:48) is complementary to divergicin A, the entire colicinV leader sequence and a region immediately upstream of the leucocin Aleader sequence. Plasmid pLD1 was made in the same way as pLED1, exceptthat MB41 (51-TGAGTAATTTTCGGTGCAGCCATAATAACTTTCCTCTTTT-3¹) (SEQ IDNO:49), a primer complementary to the region immediately upstream of theleucocin A leader sequence was used instead of MB37. In pLD1 thedivergicin A is encoded without a leader peptide. To make a fusionbetween the leucocin A leader peptide and colicin V, the leucocin Aleader sequence and the upstream region was amplified by PCR using pMJ3as template and as primers MB32 and MB43(5′-ATATCACGCCCTGAAGCACCTCCTACGACTTGTTCGA-3′) (SEQ ID NO:50) that iscomplementary to the leucocin A leader sequence and colicin V. The PCRproduct was then used as a megaprimer in a second PCR step using pHK22(Gilson et al., 1987) as a template and MB44(5-AATTAGCTTGGATCCTTCTGTGTGCATTGTCCAAT-3′) (SEQ ID NO: 51) complementaryto the downstream region of the structural colicin V gene as the reverseprimer. The resulting PCR fragment was cleaved with HindIII, arestriction site that is located in the sequence of MB44, and Sac! andcloned into pMG36e, giving plasmid pLEC1. All constructs were sequencedby the dideoxy-chain method of Sanger et al. (1977). Plasmid pTLA1 wasconstructed by cloning a 0.6 kb SacI-SspI fragment from pLAD6 thatencodes the divergicin A gene fused to the lactococcin A leader sequenceinto the multiple cloning site of plasmid pT713 (Tabor and Richardson,1985).

[0141] Overexpression of Divergicin A Precursor in E. coli by T7 RNApolymerase.

[0142] Cultures of E. coli BL21(DE3) were grown to OD600 of 0.3 in TYbroth supplemented with 0.2 mM 2,2′-dipyridyl. The cells weresubsequently induced by the addition of IPTG at a final concentration of0.4 mM. After 2 h of incubation the cells were harvested, washed andconcentrated 100-fold in deionized water, and lysed by sonication at 4°C. The lysate was applied to a tricine-SDS-polyacrylamide gel of 16%acrylamide (wt/vol) and 0.5% (wt/vol) bisacrylamide as described bySchagger and von Jagow (1987). After electrophoresis, the gel was fixedfor 30 min in 50% methanol and 10% acetic acid and washed twice with 1liter of deionized water for 1 h each. Antagonistic activity wasdetected by overlayering the gel on an APT agar plate with soft APT agarcontaining C. divergens UAL278 as the indicator strain.

[0143] Divergicin Production in Leuconostoc gelidum and Lactococcuslactis Using leader Peptides from Leucocin A and Lactococcin A.

[0144] Divergicin A is produced as a prepeptide that consists of amature peptide of 46 amino acids and a classical N-terminal signalpeptide of 29 amino acids (SEQ ID NO:6). The signal peptide ofdivergicin A was replaced with the double-glycine type leader peptidesfrom leucocin A (SEQ ID NO:9) and lactococcin A (SEQ ID NO:11) by atwo-step polymerase chain reaction (PCR) strategy as shown in FIG. 4.The DNA encoding the leucocin A leader peptide and a 176-bp upstreamregion was amplified by PCR. The resulting PCR fragment was used as amegaprimer to amplify the DNA encoding the mature peptide for divergicinand its immunity protein. The PCR product containing the gene fusion wascloned into the vector pMG36e to give plasmid pLED1. The gene fusion inpLED1 is under the control of the P32 promoter of pMG36e that isfunctional in a variety of bacteria (van der Vossen et al., 1987). Todetermine whether the secretion apparatus for leucocin A can recognizethis hybrid protein, remove the leader peptide and translocatedivergicin A into the external medium, plasmid pLED1 was introduced intoLeuconostoc gelidum UAL187-22. The genetic determinants for leucocin Aand its transport proteins LcaC and LcaD are located on one of the twoplasmids found in this organism (Hastings et al., 1991; van Belkum andStiles, 1995). Carnobacterium divergens UAL278 was used as a sensitiveindicator strain to monitor divergicin production. Because C. divergensUAL278 is sensitive to leucocin A, a strain of UAL278 that is resistantto leucocin A was isolated by exposing C. divergens UAL278 to asublethal concentration of leucocin A. This strain was used insubsequent studies to detect divergicin production. Production ofdivergicin A using this fusion construct was also monitored inLactococcus lactis IL1403 carrying plasmid pMB500. This plasmid containsgenes for the lactococcin transport proteins LcnC and LcnD and thestructural and immunity genes for lactococcins A and B (van Belkum etal. , 1989; Stoddard et al. , 1992). Lactococcins A and B are onlyactive against lactococci and do not inhibit the growth of C. divergens.When L. gelidum UAL187-22 and L. lactis IL1403(pMB500) were transformedwith pLED1, production of divergicin A was observed (FIGS. 8 and 9).However, transferring pLED1 into L. gelidum 187-13, a derivative ofUAL187-22 that has been cured of the leucocin plasmid (Hastings andStiles, 1991), or into L. lactis IL1403, production of divergicin didnot occur.

[0145] In a similar way, divergicin A was fused to the lactococcin Aleader peptide. DNA encoding the lactococcin A leader sequence and a375-bp upstream region was amplified. The resulting PCR product was usedin a second PCR reaction to fuse the lactococcin A leader sequence tothe divergicin gene. This PCR product was cloned into pMG36e, resultingin pLAD6. Transformation of pLAD6 into L. gelidum 187-22 or L. lactisIL1403(pMB500) resulted again in production of divergicin (FIGS. 8 and9). Apparently, the leucocin A and lactococcin A leader peptides candirect the secretion of divergicin using the leucocin A as well as thelactococcin A transport proteins, respectively. The data shown in FIGS.8 and 9 illustrate that L. gelidum 187-22 produced somewhat moredivergicin with pLED1 than with pLAD6, while in L. lactis IL1403(pMB500)the opposite effect was observed. This was confirmed when divergicinactivity in the supernatant of cultures of L. gelidum 187-22 and L.lactis IL1403(PMB500) transformed with these two plasmids were compared.A culture of L. gelidum 187-22 transformed with pLED1 produced fourtimes more divergicin than with pLAD6, while L. lactis IL1403(pMB500)transformed with pLAD6 doubled the production of divergicin comparedwith pLED1.

[0146] To confirm that inhibition of C. divergens UAL278 by L. gelidum187-22 carrying pLED! or pLAD6 was caused by divergicin A production andnot by leucocin A, the inhibitory compound was partially purified andthe N-terminal amino acid sequence was determined. The N-terminal aminoacid sequence of Ala-Ala-Pro-Lys-Ile from the purified peptide indicatedthat the active compound was indeed divergicin A (Worobo et al., 1995)and that proteolytic cleavage occurred at the C-terminus of the twoglycine residues of the leucocin A and lactococcin A leader peptides.This demonstrated that LcaC, the ABC transporter for leucocin A,correctly processed these leader peptides fused to divergicin A.

[0147] Some divergicin was produced when L. lactis IL1403 that did notcontain pMB500 was transformed with pLAD6 (FIG. 6). It has recently beenshown that L. lactis IL1403 carries a set of secretion genes on thechromosome that are homologous to the lactococcin secretion genes lcnCand lcnD of pMB500 (Venema et al., 1996). These results indicate thatthe transport proteins encoded on the chromosome of IL1403 recognize thehybrid protein containing the lactococcin A leader peptide but not whenit contains the leucocin A leader peptide.

[0148] Divergicin A production using the colicin V secretion apparatus.

[0149] To determine whether divergicin A fused to the leucocin A orlactococcin A leader peptides could be secreted by E. coli using thetransport proteins for colicin V, pLED1 and pLAD6 were transformed intoE. coli MC4100 carrying pHK22. Plasmid pHK22 contains the structuralgene of, and the immunity gene for, colicin V as well as the genesencoding the two inner membrane transport proteins CvaA and CvaB forcolicin V (Gilson et al., 1990). With plasmid pLED1, but not with pLAD6,divergicin could be produced in E. coli MC4100(pHK22) (FIG. 7). Tocompare the efficiency of divergicin secretion by the colicin Vsecretion apparatus using the leucocin A leader peptide with that whenthe colicin V leader peptide (SEQ ID NO:13) was used, plasmid pCOD1 wasconstructed. Plasmid pCOD1 is identical to pLED1 except that theleucocin A leader peptide was replaced by the colicin V leader peptide(FIG. 4). The zone of inhibition of C. divergens UAL278 formed by E.coli MC4100 carrying pHK22 and pCOD1 was slightly larger than thatproduced by E. coli cells carrying the two plasmids pHK22 and pLED1(FIG. 7). Divergicin production was not observed when pLED1 or pCOD1were transformed into MC4100 that did not contain pHK22. The ironchelator 2,2′-dipyridyl was used in the medium to induce the colicin Vpromoters (Chehade and Braun, 1988; Gilson et al., 1990). Omitting thisinducer from the medium greatly reduced production of colicin V as wellas divergicin A.

[0150] When L. gelidum 187-22 and L. lactis IL1403(pMB500) weretransformed with pCOD1, production of divergicin was observed inUAL187-22 but not in IL1403(pMB500) (FIGS. 8 and 9). The colicin Vleader peptide was not as efficient as the leucocin leader in directingthe secretion of divergicin in L. gelidum 187-22 (FIG. 5).

[0151] As a negative control, pLD2 was constructed. It is identical topLED1 or pCODl except that leader peptides that precede the mature partof the divergicin A peptide were excluded. E. coli MC4100 (pHK22) cellstransformed with pLD2 did not inhibit the growth of C. divergens UAL278.Furthermore, the introduction of pLED1, pLAD6 or pCOD1 into L. gelidum187-22, L. lactis IL1403(pMB500) and E. coli MC4100(pHK22) did notaffect the production of leucocin A, lactococcins and colicin V,respectively.

[0152] TolC is Required for ABC Transporter-Dependent Transport.

[0153] For translocation of colicin V across the outer membrane in E.coli, the presence of the minor outer membrane protein TolC is required(Gilson et al., 1990). To determine whether TolC is essential fordivergicin A production in E. coli, pHK22 in combination with pCOD1 orpLED1 were introduced into E. coli ZK796, a TOIC^(D) derivative ofMC4100 (Wandersman and Delepelaire, 1990). E. coli ZK796(pHK22)containing pLED1 or pCOD1 did not produce divergicin A, indicating thatdivergicin A requires the TolC protein for the ABC protein-dependentsecretion pathway in E. coli.

[0154] Colicin V Secretion in Lactococcus lactis.

[0155] The results described above indicate that leader peptides of thedouble-glycine type can direct the secretion of heterologous substratesusing ABC tranporters. To determine whether colicin V, a bacteriocin of88 amino acids (SEQ ID NO:32) that is produced by E. coli, can beexported by lactic acid bacteria using the leucocin A leader peptide,the leucocin A leader peptide was fused to colicin V. The same DNAsequence encoding the leucocin A leader peptide plus the 176-bp upstreamregion present in pLED1 was amplified by PCR and was used as amegaprimer to amplify the DNA encoding the mature part of colicin V anda downstream region of 54 bp. The resulting PCR product was cloned intopMG36e, giving plasmid pLEC2. When L. lactis IL1403(pMB500) wastransformed with pLEC2, colicin V production was observed using E. coliDH5α as the sensitive indicator strain. No inhibition was observed whenDH5α carrying pHK22 was used as the indicator strain. However,transformation of L. gelidum 187-22 with pLEC2 did not result insecretion of colicin V. Apparently, colicin V can be exported using LcnCand LcnD, but it seems that it cannot access the transport proteins forleucocin A.

[0156] The genes for the N-terminal amino acid extensions described byWorobo and associates (1995) and Quadri and associates (1994) would alsobe suitable for the using as leader sequences similar to those describedherein.

[0157] In summary this protocol can be used to generate plasmids withmore than one bacteriocin, or can be used to generate several plasmidswith different bacteriocins. Using these techniques in combination withthe nucleotide or peptide sequence of the desired leader or signalpeptide and the desired bacteriocin, one of ordinary skill in the artcan determine how to isolate the appropriate genes, identify and preparethe appropriate primers and insert the appropritate genes into a plasmidwithout undue experimentation. The host cell is the organism that issafe to use in the proposed enviroment or is responsible for aparticular function in the enviroment. For example, the particularstrain of bacteria used to make a particular type of cheese would be asuitable host for making an organism which would inhibit the growth of avariety of undesirable organisms but still make the desired type ofcheese. The desired leader sequence or signal peptide would be a leadersequence found associated with a bacteriocin derived from the samespecies of bacteria or a general signal bacteriocin peptide. Thebacteriocin selected would target undesirable organism found in theparticular enviroment. For many application such as preservation ofmeat, both Gram-negative and a Gram-positive bacteriocins are desiredtherefore two or more bacteriocins would be required (one derived from aGram-negative organism and the other derived from a Gram-positiveorganism.)

[0158] The dedicated secretion and accessory proteins of Leuconostocgelidum UAL187 can be used to produce several different bacteriocinsfrom one cell. The bacteriocins produced can be targeted against a rangeof bacteria, and those produced to date include colicin V in combinationwith one or more bacteriocin derived from leucocin A, carnobacteriocinB2 or other bacteriocins described herein.

Example 4 Spectrum of Bacteriocins Antibiotic Activity

[0159] The antibiotic spectrum of a bacteriocin can be determined by avariety of methods including but not limited to direct and deferredantagonism methods or spot-on-the lawn testing as described by Ahn andassociates (1990a and b) and van Belkum and Stiles (1995).

[0160] The spectrum of antibiotic activity of individual bacteriocinswere determined using partially purified bacteriocins. The bacteriocinswere purified by methods specific for the bacterocin (Henderson et al.1992; Hechard et al 1992; Hastings et al 1991; Quadri et al; 1993;Worobo et al. 1994; UAL-26 and Brochocin-C to be described later) orobtained commercially such as Pediocin PA-1 (Quest; Flavors & FoodIngredients Co., Rochester, N.Y.). Bacteriocins activity was determinedusing Carnobacterium divergens LV13 grown on ATP agar and expressed inarbitrary units of inhibitory activity (AU) based on the reciprocal ofthe greatest dilution that is inhibitory to this indicator strain (Ahnand Stiles 1990). Several bacteriocins were tested using 10 μl/spot of100 AU/ml or 800 AU/ml for inhibtion of growth of a variety of strainsof bacteria grown on agar (APT for most organisms except for thefollowing: Lactobacilli MRS broth containing 1.5% agar for Lactobacillusand Pediococcus strains; Tryptic Soy Broth containing 1.5% agar (TSBagar) for Bacillus, Staphylococcus and Streptococcus strains; TSB plus0.6% yeast extract for Listeria strains; or Trypticase Peptone GlucoseYeast extract for Clostridium strains and the results are summarized inTables 2, 3 and 4.

[0161] This procedure can be used to test the ability of specificbacteriocins to inhibit the growth of specific organisms. With thisinformation partially purified or purified bacteriocins can beidentified for the use in the control of the growth of particularorganisms, particular groups of organisms or for the treatment ofparticular diseases.

[0162] Organisms can be engineered as described herein to incorporateone or more of the desired bacteriocins for the inhibition of the growthof particular organisms or groups of organisms using the geneticallyengineered organism.

[0163] Carnobacteriocin 26, Enterocin 900 and Brochocin-C would be verygood inhibitors of a broad range organisms as indicated in Table 2, 3and 4. Inhibition of the growth of these organisms is important fordisease control or to reduce spoilage of agricultural products.

Example 5 Molecular Characterization of Genes Involved in the Productionof the Bacteriocin Leucocin A from Leuconostoc gelidum

[0164] Leucocin A is a bacteriocin produced by Leuconostoc gelidumUAL187 isolated from vacuum packaged meat (Hasting and Stiles; 1991). Itinhibits a wide spectrum of LAB as well as some strains of Listeriamonocytogenes and Enterococcus faecalis. Curing experiments of UAL187showed that the genetic determinant for leucocin A was located on one ofthe three plasmids found in this organism. The bacteriocin was purifiedand shown to contain 37 amino acids (Hastings et al. 1991). A degenerateoligonucleotide was used for hybridization with plasmid DNA of UAL187-22which has only two of the three plasmids, pLG7.6 and pLG9.2, and stillproduces bacteriocin (Hastings and Stiles 1991). A 2.9-kb HpaII fragmentof pLG7.6 showing homology was cloned and sequenced revealing thestructural gene for leucocin A (lcnA) and a second open reading frame(ORF). It was postulated that this second ORF could encode an immunityprotein (Hastings et al. 1991). Leucocin A was shown to be produced as aprecursor with a 24 amino acid N-terminal extension. Transformation ofseveral LAB with constructs containing the 2.9-kb fragment did not showproduction of leucocin A. UAL187-13, a cured, bacteriocin-negativederivative of the wild type strain, was refractory to transformation.

[0165] Leucocin A is a small heat stable bacteriocin produced byLeuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA thatcontains the leucocin structural gene and a second open reading frame(ORF) in an operon was previously cloned (Hastings, et al. 1991). When a1-kb DraI-HpaI fragment containing this operon was introduced into abacteriocin-negative variant (UAL187-13), immunity but no leucocinproduction was detected. Leucocin production was observed when an 8-kbSacI-HindIII fragment of the leucocin plasmid was introduced into Leuc.gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequenceanalysis of this 8-kb fragment revealed the presence of three ORFs in anoperon upstream and on the opposite strand of the leucocin structuralgene. The first ORF (lcaE) encodes a putative protein of 149 aminoacids. The second ORF (lcaC) contains 717 codons and encodes a proteinthat is homologous to members of the HlyB-family of ATP-dependentmembrane translocators. The third ORF (lcaD) contains 457 codons thatencodes a protein with strong resemblance to LcnD, a protein essentialfor the expression of the lactococcal bacteriocin lactococcin A.Deletion mutations in lcaC and lcaD resulted in loss of leucocinproduction, indicating that LcaC and LcaD are involved in thetranslocation and production of leucocin A. A mutation in lcaS did notaffect leucocin production. The secretion apparatus for lactococcin Adid not complement mutations in the lcaCD operon to express leucocin Ain L. lactis, but lactococcin A production was observed when thestructural and immunity genes for this bacteriocin were introduced intoa leucocin producer of Leuc. gelidum UAL187, indicating that lactococcinA could access the leucocin A secretion machinery.

[0166] To prevent confusion with nomenclature used for the genesinvolved in the expression of lactococcins, lcaA and ORF2 (Hastings etal. 1991) have been renamed lcaA and lcaB, respectively. We report thecloning and nucleotide sequence analysis of a second operon which islocated adjacent to, and on the opposite strand of, the lcaAB operon. Aconstruct containing the two operons was successfully transferred intoLeuc. gelidum UAL187-13 and resulted in leucocin production. Bacterialstrains, olasmids and media. The bacterial strains and plasmids used inthis study are listed in Table 1. Escherichia coli was grown in TY broth(Rotlander and Trautner, 1970) at 37° C.; L. lactis was grown inGlucose-M17 broth (Terzaghi and Sandine 1975) at 30° C.; and Leuc.gelidum and Carnobacterium piscicola were grown in APT broth (AllPurpose Tween; Difco Laboratories Inc., Detroit, Mich.) at 25° C. Brothmedia were supplemented with 1.2% (wt/vol) agar for solid plating media.Selective concentrations of erythromycin for growth of E. coli, L.lactis and Leuc. gelidum containing recombinant plasmids were 200, 5 and5 mg/ml, respectively. When appropriate, ampicillin was used at a finalconcentration of 150 mg/ml for E. coli, and kanamycin was used at afinal concentration of 50 mg/ml for L. lactis

[0167] Bacteriocin Assay.

[0168] To test for production of leucocin, cells of L gelidum or L.lactis were inoculated, unless otherwise stated, onto APT andglucose-Ml7 agar plates, respectively, and incubated at 25° C. for 18 h.Soft APT agar (0.7% [(wt/vol]) containing C. piscicola LV17C as theindicator strain was then poured onto the surface. After 15 h ofincubation, the plates were examined for zones of inhibition. Immunityor resistance of the different strains to leucocin was determined by aspot-on-lawn test of 0.5 μg of the bacteriocin (Ahn & Stiles, 1990).Lactococcin production was tested as described above with L. lactisIL1403 as the indicator strain in soft glucose-M17 agar (0.7% [wt/vol]).

[0169] Molecular Cloning.

[0170] Plasmids from E. coli were isolated by the method described byBirnboim and Doly (1979). With some modifications, the same method wasused to isolate plasmids from L. gelidum and L. lactis. Cells were lysedat 37° C. in 50 mM Tris-HCl (pH 8) −10 mM EDTA containing 5 mg oflysolzyme and 100 ug of mutanolysin (Sigma. St. Louis, Mo.) per ml for20 min. Restriction endonucleases, the Klenow fragment of the E.coli DNApolymerase I, and T4 DNA ligase were obtained from Promega (Madison,Wis.). Bethesda Research Laboratories (Burlington, Ontario, Canada),Boehringer Mannheim (Dorval, Quebec, Canada), or New England Biolabs(Mississauga, Ontario, Canada), and used as recommended by the supplier.Cloning and DNA manipulations were performed as described by Sambrook etal. (Sambrook et al.,1989). Competent E.coli cells were transformed bythe method of Mandel and Higa (Mandel & Higa, 1970). Transformation ofL. lactis by electroporation was performed with a Bio-Rad gene pulser(Bio-Rad Laboratories. Richmond, Calif.) by the method of Holo and Nes(Holo & Nes 1989). For transformation of L. gelidum cells werecultivated in APT broth supplemented with 3% (wt/vol) glycine.Exponentially growing cells were harvested, washed once with water andtwice with ice-cold electroporation buffer (5 mM potassium phosphatebuffer [pH 7], 3 mM MgCl₂, in 1 M sucrose), and concentrated 100-fold inthe same buffer. Subsequently, 50 μl of the cell suspension was mixedwith 2 al of plasmid DNA and held on ice for 5 min prior toelectroporation. Immediately after electroporation, cells were dilutedin 1 ml of APT containing 0.5M sucrose and 20 mM MgCl₂ and incubated for3 h at 25° C. Cells were plated on APT agar containing the appropriateantibiotic, and transformants were visible after 3 to 4 days ofincubation at 25° C.

[0171] Southern Hybridization.

[0172] For Southern hybridization, DNA was transferred to Hybond N(Amersham Canada Ltd., Oakville, Ontario,Canada), as described bySambrook et al (Sambrook et al, 1989). Nonradioactive DNA probes weremade with a random-primed labeling and detection kit (BoehringerMannheim). Hybridization and immunological detection were performed asrecommended by the supplier.

[0173] DNA Sequencing.

[0174] Nucleotide sequence analysis was performed by sequencing the DNAin both orientations by the dideoxy-chain method of Sanger et al.(Sanger et al.,1977). DNA was sequenced by Taq Dye Deoxy Cyclesequencing on an Applied Biosystems 373A DNA sequencer (AppliedBiosystems, Foster City, Calif.). for sequencing, stepwise deletionderivatives of cloned DNA fragments were made with the Erase-a-Basesystem from Promega. In addition, a primer-walking strategy was used fornucleotide sequencing. Synthetic oligonucleotides were make with anApplied Biosystmens 391 PCR-Mate DNA sythesizer. Analysis of thenucleotide sequence was performed with a software program (DNASTAR,Inc., Madison, Wis.) The search for homology of the predicted amino acidsequences with those of proteins in the Swiss-Prot protein sequencedatabase (release 30) was based on the FASTA algorithm of Pearson andLipman (Pearson & Lipman, 1988).

[0175] Nucleotide Sequence Accession Number.

[0176] The entire nucleotide sequence is sequence number is presented inthe paper van Belkum and Stiles, 1995, and some important sections ofthis gene are included in SEQ ID NO:3 (accession number L40491).Leuconostoc gelidum (strain UAL187) leucocin A ATP-dependent transporterand secretory nucleotide sequence herein referred to as SEQ ID NO:4.This sequence if incorporated into a vector and used to transform a cellenables a cell to export polypeptides with an a variety of N-terminalleader peptides including but not limited to polypeptides with aLeucocin A or a Colicin V leader peptide. Both the ABC-transporter(lcaC) herein referred to as SEQ ID NO:4 and accessory protein (lcaD)genes herein referred to as SEQ ID NO:5 are required for a functionaltransport pathway.

[0177] Cloning of the Genes Involved in the Production of Leucocin A.

[0178] The 2.9-kb HpaII fragment containing the lcaAB operon was clonedin pUC118, resulting in pJH6.1F, and in the shuttle vector pNZ19 to formthe plasmid pJH8.6L. Attempts to transform Leuc. gelidum UAL187-13 withpJH8.6L were unsuccessful (Hastings et al. 1991). Therefore, we used adifferent vector to introduce the 2.9-kb fragment (FIG. 2) into strainUAL187-13. Using the multiple cloning site of pUC118, the 2.9-kb insertin plasmid pJH6.1F was excised by digestion with EcoRI and HindIII andcloned into the EcoRI-HindIII sites of pGKV210. The resulting plasmid,pMJ1, was used to transform strain UAL187-13. However, all of thetransformants examined showed the presence of spontaneous deletionderivatives of pMJ1. When a 1-kb DraI-HpaI fragment containing lcaA andicaB was subcloned from the 2.9-kb fragment into the SmaI site ofpGKV210, the resulting recombinant plasmid pMJ3 (FIG. 2) formed a stabletransformant in Leuc. gelidum UAL187-13. This transformant was immune toleucocin A but did not produce the bacteriocin. Apparently, additionalinformation encoded on pLG7.6 is required for expression of thebacteriocin phenotype. The plasmid pMJ20 (FIG. 2) was constructed byintroducing a frame shift mutation in lcaB, by filling in the uniqueClaI site with Klenow DNA polymerase. Immunity was not observed inUAL187-13 carrying this plasmid, indicating that lcab encodes theprotein necessary for immunity to leucocin A.

[0179] Because additional genetic information is required for leucocin Aproduction, regions adjacent to-the lcaAB operon (FIG. 2) were cloned.It was previously reported that the producer strain UAL187-22 containsplasmids pLG7.6 and pLG9.2 of 7.6 and 9.2 MDa, respectively (Hastingsand Stiles, 1991). Restriction analysis of plasmid DNA from UAL187-22revealed that the actual sizes of pLG7.6 and pLG9.2 were 18 and 21 kb,respectively. To localize the lcaAB genes, Southern analysis of plasmidDNA with the 1-kb DraI-HpaI fragment as probe detected a 12.3 kb HindIIIfragment that was cloned into pUC118 to give pMJ4. Subclones of thisfragment into a shuttle vector gave pMJ6 and pMJ10 (FIG. 2).

[0180] Plasmids pMJ6 and pMJ10 were transformed into L. lactis IL1403and screened for leucocin A production. Transformants containing pMJ6but not pMJ10 inhibited the growth of the indicator strain C. piscicolaLV17C. However, the zones of inhibition of these transformants wereclearly smaller than those formed by Leuc. gelidum 187-22 (FIG. 3A). L.lactis has natural resistance to leucocin, therefore, the immunityphenotype to leucocin A could not be detected in L. lactis.Transformation of the bacteriocin-negative strain Leuc. gelidumUAL187-13 with pMJ6 resulted in several transformants containingdeletion derivatives of pMJ6 that did not show production of thebacteriocin. A transformant of UAL187-13 which contained a plasmid withthe expected size and restriction pattern of pMJ6 produced a zone ofinhibition comparable to that formed by UAL187-22 (FIG. 3A). Theseresults indicate that the genes responsible for the production ofleucocin A are located on an 8-kb SacI-HindIII fragment of pLG7.6.

[0181] Nucleotide Sequence Analysis.

[0182] Restriction analysis of pMJ6 revealed the location andorientation of the lcaAB operon on the 8-kb fragment (FIG. 2). Thenucleotide sequence of the region upstream of the lcaAB operon wasdetermined in both directions by the dideoxy-chain termination method.The nucleotide sequence in van Belkum and Stiles paper (1995) and partlyin SEQ ID NO:3 shows a 4.3-kb segment located adjacent to the previouslyreported nucleotide sequence containing the lcaAB operon as well as partof this previously reported nucleotide sequence (Hastings et al. 1991).The start of an open reading frame (ORF) was identified 151 bases from,and on the opposite strand to, the start codon of lcaA. This ORF,designated lcaE, could encode a protein of 149 amino acids and isfollowed by a TAA stop codon. Immediately downstream of lcaf, a secondORF (lcaC) was found that contained 717 codons. The TAA stop codon oflcaC is immediately followed by an ORF that could encode a protein of457 amino acids and has a TAG stop codon. All three ORFs are preceded byprobable ribosomal binding sites. A possible promoter sequence was foundupstream of lcaE (van Belkum and Stiles , 1995). However, a putativepromoter sequnce was also found within the lcaE gene. The sequence ofits −35 (TGGACT) and −10 (TACAAT) regions closely resembles theconsensus sequence of constitutive promoters found in other LAB (van deGuchte et al. 1992). The spacing of 16 and 19 bases between the −35 and-10 regions of these promotor sequences agrees with that of the usualspacing found in LAB promoters. An imperfect inverted repeat was found 6bases downstream of the stop codon of lcaD which has the characteristicsof a possible rho-independent terminator. No other ORFs and palindromicstructures were found in either strand in this 4.3-kb region.

[0183] Similarity of LcaC and LcaD to Bacterial Transport Proteins.

[0184] The hydrophobicity plot of the putative LcaC protein revealedthat the N-terminal region contains several hydrophobic domains. Ahomology search with other amino acid sequences in the SwissProtdatabase showed that LcaC belongs to the HlyB-like family of ABCtransporters (Blight and Holland 1990; Higgins 1992). These proteinscontain a highly conserved ATP binding domain in the C-terminal regionand several membrane spanning domains in the N-terminal half of thesequence. Homology comparison of HlyB, which is involved in thesecretion of hemolysin A, and LcaC revealed that 58% of the amino acidswere similar when conserved residue substitutions are included and 27%were identical. However, LcaC has a much higher degree of homology withseveral other ABC transporters. ComA, a protein from Streptococcuspneumoniae that is required for competence induction for genetictransformation (Hui and Morrison 1991) shares 59% identity and 82%similarity with LcaC. Comparison of LcaC with LcnC, a protein that isimplicated in the secretion of the lactococcal bacteriocin lactococcin Aand possibly in the secretion of lactococcins B and M (Stoddard et al.1992, van Belkum 1994), and PedD, which is involved in the production ofpediocin PA-1 (Marugg et al. 1992), revealed 81% similarity and 57%identity, and 73% similarity and 51% identity at the amino acid level,respectively. The databank search showed further that LcaC was veryhomologous to SapT (82% similarity, 57% identity) and SapT (81%similarity, 58% identity), proteins that are encoded by DNA sequenceslinked to sakacin A and 2, respectively. The highest score however, wasfound with MesC, a protein encoded in a DNA sequence linked tomesentericin Y that was nearly identical to LcaC with 99% similarity and98% identity.

[0185] Analysis of the hydropathy profile of LcaD showed a largelyhydrophilic protein with the exception of a strong hydrophobic region atthe N-terminus. Homology search in the data bank revealed that LcaD issimilar to LcnD, another protein that is essential for lactococcinproduction in L. lactis (Stoddard et al. 1992). LcaD showed 35% identityand 54% similarity to LcnD. Additional homologues of LcaD that werefound were SspE (62% similarity, 32% identity), SapE (65% similarity,35% identity) and MesY (96% similarity, 87% identity) whose genes arelinked to the genetic determinants for sakacin A, P and mesentericin Y,respectively. Two other proteins that showed similarity with the LcaDprotein were ComB from S. pneumoniae (Hui et al. 1995) with 61%similarity and 29% identity and the ORF1 protein encoded byLactobacillus johnsonii (Fremaux et al. 1993). The ORF1 protein hassimilarity with the N- and C-termini of LcaD. The ORF1 protein isencoded by a 5′ end truncated ORF of 540 bases located upstream of thebacteriocin operon responsible for lactacin F production (Fremaux et al.1993).

[0186] The hydropathy profile of the putative protein LcaE showed arather hydrophilic protein. Search of the databank revealed onlysimilarity of LcaE to MesE, a protein encoded by a DNA sequence linkedto mesentericin Y production.

[0187] Functional and Complementation Analyses of LcaC and LcaD.

[0188] To establish whether lcaE, lcaC and lcad are essential forleucocin production, deletion and mutation derivatives of pMJ6 wereconstructed in E. coli (FIG. 2). Deletion of the BstEII-StuI fragment inlcaC resulted in plasmid pMJ17. Cells of Leuc. gelidum UAL187-13containing this construct were immune to leucocin but bacteriocin wasnot produced. If we assume that the deletion had no polar effect onlcad, the result would indicate that lcaC is involved in leucocinproduction. Two deletion constructs in lcaD were made, namely pMJ16 andpMJ18. In plasmid pMJl6 an EcoRV-BamHI fragment was deleted, whereas anEcoRV-HindIII fragment was deleted in pMJ18. A frame shift mutation inlcae was made using the NsiI restriction site, giving plasmid pMJ26.Several attempts to introduce pMJ16, pMJ18 and pMJ26 into UAL187-13 wereunsuccessful. When pMJ16 and pMJ17 were introduced into L. lactisIL1403, bacteriocin production was not detected. However, transformationof L. lactis IL1403 with pMJ26 did not affect leucocin production. Theseresults indicate that LcaD, but not LcaE, is essential for leucocinproduction. Given the high degree of similarity between LcaC and LcaD ofLeuc. gelidum and LcnC and LcnD of L. lactis, it was decided todetermine whether the mutations in lcaC and lcaD could be complementedby the lactococcin A gene cluster in L. lactis IL1403 carrying pMB500(Stoddard et al. 1992; van Belkum et al 1989). Plasmids pMJ3, pMJ16 andpMJ17 were used to transform IL1403(pMB500). Although the differentplasmids contain the same replicon as pMB500, transformants can beselected for erythromycin resistance and pMB500 can be selectivelyretained by its own lactococcin production and resistance to kanamycin.However, leucocin production was not observed in these transformants,indicating that proper complementation by the lactococcin secretionapparatus was not possible. Only transformation of IL1403 (pMB500) withpMJ6 resulted in a zone of inhibition. In contrast, transformation ofLeuc. gelidum UAL187-22 with plasmid pMB553, which carries thestructural and immunity genes for lactococcin A showed a small zone ofinhibition using L. lactis IL1403 as an indicator (FIG. 3B). LactococcinA is only active against lactococci (Holo et al. 1991). No such zone ofinhibition was observed when UAL187-13 was transformed with pMB553.Apparently, the leucocin secretion system is able to complement the lcnCand lcnD genes for the secretion of lactococcin A to a limited extent.

Example 6 Novel Bacteriocin Nucleotide and Amino Acid Sequences(Brochocin-C)

[0189]Brochothrix campestris ATCC 43754 isolated from soil as reportedby Siragusa and Nettles Cutter ( ) to produce a broad spectrumbacteriocin. They did not characterize the bacteriocin and did not showthat it is active against C. botulinum. We have now demonstrated thatthis is a two-component bacteriocin that is chromosomally produced andthat the translation products of the two genes responsible for activityand an immunity gene (FIGS. 13, 14, 15 and 16).

[0190] Biochemical and Aenetic Characterization of Brochocin-C.

[0191] Brochocin-C is a strongly hydrophobic bacteriocin produced byBrochothrix campestris ATCC 43754 that is active against a broadspectrum of Gram-positive bacteria (Table 2 and 3). Crude brochocin-Cwas thermostable up to 121° C. for 15 min, pH stable from 2 to 9, andinactivated by proteolytic enzymes. The bacteriocin was purified, itsnucleotide (SEQ ID NO:21) and amino acid sequence determined, and asite-specific 23-mer oligonucleotide probe was synthesized whichhybridized to a 4.2-kb EcoRI genomic DNA fragment. The two components ofthe bacteriocin, brochocin A (nucleotide sequence herein referred to asSEQ ID NO:22 and amino acid sequence herein referred to as SEQ ID NO:23)and B (nucleotide sequence herein referred to as SEQ ID NO:24 and aminoacid sequence herein referred to as SEQ ID NO:25), and their immunitygene (nucleotide sequence herein referred to as SEQ ID NO:26 and aminoacid sequence herein referred to as SEQ ID NO:27) have been clonedseparately and fused behind the signal peptide of divergicin A andproduced in different hosts. Both Brochocin A and B contain a N-terminalleader peptide that gets cleaved after a double glycine motif to yieldmature a bacteriocin and a leader peptide. This leader peptide bearssignificant homology to leader peptides of the class II bacteriocins oflactic acid bacteria.

[0192] Bacterial Strains and Plasmids:

[0193] The bacterial strains and plasmids used in these studies arelisted in Table 8. These include strains from the American Type CultureCollection (ATCC), Brochothrix strains from G. G. Greer isolated frommeat at the Lacombe Research Centre and from our laboratory culturecollection (UAL). All strains with the exception of Escherichia coliwere stored at −70° C. in All Purpose Tween (APT) broth (DifcoLaboratories Inc., Michigan) adjusted to pH 6.5, supplemented with 20%glycerol (v/v). Cultures for use in experimental studies were obtainedby inoculation of frozen cells into APT broth at pH 6.5, and subculturedfor two successive transfers at 25° C. after 18 to 24 h before beingused. Growth experiments and (or) bacteriocin production from B.campestris were done in APT broth, Cooked Meat Medium (CMM; Difco), orsemi-defined casamino acids medium (CAA), described by Hastings et al.(1991). CAA medium was used for the purification of the bacteriocin.

[0194]E. coli strains were stored at −70° C. in Luria-Bertani (LB) broth(Sambrook et al. 1989) supplemented with 40% glycerol (v/v). Inoculationof E. coli strains was done from frozen cultures into LB broth withampicillin or erythromycin added to a final concentration of 200 mg/mLand propagated at 37° C. with shaking (250 rpm). Potential pUC118recombinants were identified by the blue-white colour selection fromgrowth on LB plates (1.5% w/v granulated agar) supplemented withampicillin (200 mg/mL) and used with X-gal.(5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) and IPTG(isopropylthio-β-D-galactopyranoside) TABLE 8 Bacterial strains andplasmids Organism Reference Bacillus macerans ATCC 7048 ATCC B. cereusATCC 14579 ATCC Brochothrix campestris ATCC 43754 ATCC B. campestris MTThis study B. thermosphacta ATCC 11509 ATCC B. thermosphacta I41 UAL B.thermosphacta B1-B5, B7-B16 (inclusive) GGG Carnobacterium piscicolaLV17 Shaw C. piscicola LV17A Ahn and Stiles (1990n) C. piscicola LV17BAhn and Stiles (1990b) C. piscicola LV17C Ahn and Stiles (1990b) C.piscicola C2/8B Quadri et al. (1994) C. piscicola C2/8A Quadri et al.(1994) C. piscicola UAL26 Burns (1987) C. piscicola UAL26/8A Ahn andStiles (1990b) C. piscicola UAL26/8B Quadri et al. (1994) C. divergensLV13 Shaw C. divergens 9/8A Quadri et al. (1994) C. divergens 9/8BQuadri et al. (1994) Clostridium bifermentans ATCC 19299 ATCC C.butyricum ATCC 8260 ATCC C. pasteurianum ATCC 6013 ATCC Enterococcusfaecalis ATCC 19433 ATCC E. faecalis ATCC 7080 ATCC E. faecium ATCC19434 ATCC E. durans ATCC 11576 ATCC Lactobacillus sake Lb706Schillinger L. plantarum ATCC 4008 ATCC Lactococcus lactis ATCC 11454ATCC L. lactis UAL 245 UAL L. lactis UAL 276 UAL Leuconostoc gelidum UAL187 Hastings et al. (1991) L. gelidum UAL 187.13 Hastings et al. (1991)L. gelidum UAL 187.22 Hastings et al. (1991) L. mesenteroides ATCC 23386ATCC L. mesenteroides Y105 Cenatiempo Listeria innocua ATCC 33090 ATCCL. monocytogenes Scott A ATCC L. monocytogenes I42 UAL L. monocytogenesATCC 15313 ATCC Pediococcus acidilactici ATCC 8042 ATCC P. acidilacticiPAC 1.0 Vandenbergh Staphylococcus aureus S6 HPB S. aureus S13 HPBEscherichia coli DH5α BRL Laboratories Life Technologies Inc. E. coliAP4.7 (DH5α containing pAP4.7) This study E. coli AP7.4-32 (DH5αcontaining pAP7.4) This study E. coli AP4.6-8 (DH5α containing pAP4.6)This study Plasmids pUC118 (3.2 kb; Amp^(R); lac Z′ ) Vieira andMessing, (1982) pGKV210 (4.4 kb; Em^(R)) van der Vossen et al. (1985)pAP4.7 (pUC118; 1.6 kb EcoRI - This study PstI fragment) pAP7.4 (pUC118;4.2 kb EcoRI This study fragment) pAP4.6 (pUC118; 1.4 kb PstI This studyfragment) pAP8.6 (pGKV210; 4.2 kb EcoRI This study fragment)

[0195] ATCC=American Type Culture Collection

[0196] BRL=Bethesda Research Laboratories Life Technologies Inc.

[0197] UAL=University of Alberta Food Microbiology culture collection

[0198] GGG=G. Gordon Greer (Lacombe Research Centre, Alberta, Canada)

[0199] HPB=Health Protection Branch (Ottawa, Ontario, Canada)

[0200] Shaw=B. G. Shaw (AFRC Institute of Food Research, Bristol, UK)

[0201] Vandenbergh=P. A. Vandenbergh (Quest International, Sarasota, US)

[0202] Burns=K. Burns (M.Sc. thesis, 1987, University of Alberta,Edmonton, AB)

[0203] Schillinger=U. Schillinger (Institute of Hygiene and Toxicology,Federal Research Centre for Nutrition, Karlsruhe, Germany)

[0204] Cenatiempo=Y. Cenatiempo (Institut de Biologie Moléculaire etd'Ingénierie Génétique, Centre National de la Recherche Scientifique,Université de Poitiers, France)

[0205] at final concentrations of each at 1.6 mg/mL.Erythromycin-resistant (Em^(R)) transformants of E. coli with pGKV210were selected on either LB or YT (yeast extract, tryptone; Difco) agarwith erythromycin (200 mg/mL ).

[0206] Bacteriocin Assays.

[0207] Antagonistic bacteriocin activity against different indicatorstrains was determined by direct or deferred inhibition assays (Ahn andStiles, 1990b). For direct inhibition tests, broth cultures wereinoculated onto APT agar (1.5%) plates using a Cathra replicator,allowed to dry, and immediately overlayered with 7.5 mL of molten APTagar (0.75% agar) at 45° C., seeded with a 1% inoculum of the indicatorstrain. For deferred inhibition tests, inoculated cells were incubatedat 25° C. for 15 to 18 h before being overlayered with the indicatorstrain as described above. In both instances, overlayered plates wereplaced in an anaerobic jar (BBL) filled with a 10% Co₂ and 90% N₂atmosphere and incubated at 25° C. for 16 to 24 h before analyzing theresults.

[0208] Bacteriocin activity of B. campestris ATCC 43754 was detected orquantified by the spot-on-lawn method (Ahn and Stiles, 1990b) against C.piscicola LV17C. Doubling dilutions (1:1) of cell supernatants (heattreated at 65° C. for 30 min) were prepared in sterile water and 10 or20 mL of each dilution was spotted onto an APT plate freshly overlayeredwith the indicator lawn. Activity was determined by taking thereciprocal of the highest dilution which showed a distinct zone ofinhibition of the indicator strain, and expressed as arbitrary activityunits (AU) per mL.

[0209] Stability of Brochocin-C.

[0210] The effects of pH and heat treatment on the activity of crudebrochocin-C were determined. Cultures grown in APT broth werecentrifuged (8000× g for 15 min) and the supernatant was adjusted to pH2 through 9 using either 5 N HCl or NaOH. The pH-adjusted supernatantwas heated at 65° C. for 30 minutes before doing a spot-on-lawn assay.Heat stability of brochocin-C in pH-adjusted supernatant was determinedby heating at 65° C. for 30 min, 100° C. for −15 min, or 121° C. for 15min before testing the residual activity of each sample and comparing itwith the activity in unheated supernatant. To test the effect of organicsolvents on the activity of brochocin-C, preparations of brochocin-Cpartially purified by butanol extraction (see below) were diluted ineither 0.1% trifluoroacetic acid (TFA), 95% ethanol, 100% methanol, or100% acetonitrile to give an initial concentration of 10 AU/mL. Tubeswere incubated at 25 and 4° C. for selected time intervals before a 10mL aliquot of each treatment was removed and spotted onto a freshlyoverlayered lawn of C. piscicola LV17C. Sizes of the zones of inhibitionwere measured and compared to that at time zero for each treatment.

[0211] Plasmid Curing.

[0212] Overnight cultures of B. campestris were inoculated at 10⁷ cfu/mLinto APT broth containing different concentrations of the curing agentsnovobiocin, acriflavin, and sodium-dodecyl sulphate (SDS) and grown at25° C. for 24 h to determine the minimum inhibitory concentration ofeach.

[0213] The loss of bacteriocin production was determined from culturesgrown in acriflavin by heating a 500 mL aliquot of the culture at 65° C.for 30 min and spotting it onto a lawn of C. piscicola LV17C. A negativecontrol of sterile APT broth with the different concentrations ofacriflavin was also spotted onto the indicator lawn to ensure that theacriflavin did not have an inhibitory effect on the indicator cells.Curing was attempted using a combination of acriflavin and elevatedgrowth temperature (30° C.) using an inoculum of 10⁴ cfu/mL in APT brothwith the selected acriflavin concentration. The culture was grown untilturbidity was detected and then it was subcultured an additional 1 to 6times at inocula of 10³ or 10⁴ cfu/mL in APT broth containing the sameacriflavin concentrations. Dilutions of these cultures were made insterile 0.1% peptone (Difco) water and plated onto APT plates. Plateswere incubated in anaerobic jars at 25° C. for 2 d and replica-platedonto two other APT plates, allowed to grow for 2 d before overlayeringone plate with C. piscicola LV17C and the other with Listeriamonocytogenes 33090. Colonies showing a loss of bacteriocin productionwith both of the indicator strains were inoculated into APT broth forsmall-scale plasmid isolation (see below). The wild-type strain was alsoincluded in the small-scale plasmid isolations to serve as a positivecontrol.

[0214] Purification of Brochocin-C.

[0215] A flask containing five liters of sterile CAA medium (Hastings etal., 1991) with 2.5% glucose was inoculated with 2% of an overnightculture of B. campestris ATCC 43754, and grown at a constant pH of 6.7with a Chemcadet (Cole-Parmer, Chicago, Ill.) by addition offilter-sterilized (0.22 mm) 2 M NaOH. Growth of the culture wasmonitored and stopped after 22 h of incubation at 25° C. Cells wereremoved from the culture broth by centrifugation at 8000× g for 20 min.The supernatant (approximately 5.5 liters) was extracted twice with 1.5liters of n-butanol. The extract was diluted with water (approximately1:1), concentrated on a vacuum evaporator at 35° C. and evaporatedrepeatedly to remove the last traces of butanol. The extract wassuspended in water (approximately 150 mL), precipitated with 1.7 litersof cold (−60° C.) acetone and stored at 5° C. for 24 h. The precipitatewas separated by centrifugation (10,000× g for 15 min), dissolved in 10mL of 0.1% TFA and loaded onto a Sephadex G50 (Pharmacia) column(2.5×120 cm) that had been pre-equilibrated with 0.1% TFA. The columnwas washed with 0.1% TFA at a flow rate of approximately 0.6 mL/min.Absorbance of collected fractions was monitored at 220 nm. Fractionsshowing antimicrobial activity by spot-on-lawn assay were concentratedand lyophilized. The purity of the sample was confirmed by mass spectrumanalysis and sodium-dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE).

[0216] SDS-PAGE.

[0217] Brochocin-C preparations were examined on 20% poly-acrylamidegels with the buffer system described by Laemmli (1970) in 3M Tris-HCl.Electrophoresis was done at 20 mA constant current for 3 h. Afterelectrophoresis, gels were fixed in 50% methanol, 10% acetic acid for 1h and stained with Coomassie blue (Bio-Rad) or assayed for antimicrobialactivity by overlayering with C. piscicola LV17C as the indicator strainby the method of Barefoot and Klaenhammer (1983).

[0218] Inhibition by Brochocin-C.

[0219] Partially purified preparations of brochocin-C were obtained bybutanol extraction of supernatant fluids of an overnight culture of B.campestris ATCC 43754 grown in CAA medium with constant pH regulation at6.7. All traces of butanol were removed by rotary evaporation. Thepartially purified bacteriocin was added to APT broth (pH 6.5) and tophosphate buffer (50 mM, pH 7.0) containing 106 cfu/mL of C. piscicolaLV17C. The bacteriocin was added to give a final concentration of 100AU/mL and the tubes were incubated at 25° C. Viable counts weredetermined by enumeration on APT agar at selected time intervals andcell lysis was checked by monitoring the optical density at 600 nm. Forenumeration, cultures grown in APT broth and phosphate buffer werediluted in sterile 0.1% peptone water and 50 mM phosphate buffer (pH7.0) respectively. Growth of the indicator strain without addition ofbacteriocin was also included as a control.

[0220] Determination of the Amino Acid Sequence and the Amino AcidContent of Brochocin-C.

[0221] The N-terminal amino acid sequence of brochocin-C was determinedby automated Edman degradation with a gas-phase sequencer (AppliedBiosystems model 470A) with on-line phenylthiohydantoin-derivativeidentification by HPLC (Applied Biosystems model 120A chromatograph).The amino acid content of purified brochocin-C was determined byderivitization with phenylisothiocyanate on an Applied Biosystems 420Aderivatizer and separation with a C₁₈ column by HPLC (Applied Biosystemsmodel 130A chromatograph)

[0222] The mass spectrum of purified brochocin-C was measured by plasmadesorption and fast atom bombardment (FAB).

[0223] DNA Isolation, Manipulation, and Hybridization.

[0224] Small-scale plasmid isolation of B. campestris was done bypreviously established methods (Ahn and Stiles, 1990b). Cells from anovernight culture grown in APT broth were recovered by centrifugation at14,000× g for 5 min, washed once with cold 0.5% NaCl (500 mL), andresuspended in 100 mL of solution A (25% sucrose, 50 mM Tris-HCl, 5 mMEDTA, pH 8.0) containing lysozyme (10 mg/mL). After incubation for 1 hat 37° C., 200 mL of solution C (0.9% glucose, 3% SDS, 50 mM Tris-HCl, 5mM EDTA, pH 8.0) containing 0.2 N NaOH was added and the tubes weregently inverted several times until the cell lysate cleared. Solutionsof 2 M Tris-HCl, pH 7.0 (50 mL) and 5 M NaCl (70 mL) were added to thetubes and mixed by inversion. The DNA was extracted once with 3%NaCl-saturated phenol/chloroform and once withchloroform/isoamyl-alcohol (24:1), before overnight precipitation at−20° C. with 95% ethanol. Large-scale preparation of plasmid DNA wasdone by scaling up (100×) of the small-scale method using cells from 750mL of an overnight culture grown in APT broth and purified byCsCl—ethidium bromide density gradient ultracentrifugation. The CsClsalt was removed by dialysis in 10 mM Tris-HCl, 1 mM EDTA (TE buffer, pH8.0; Sambrook et al., 1989). Chromosomal DNA preparation of B.campestris was done as described by Quadri et al. (1994), but wasresuspended in a final volume of 1 mL TE buffer. An equal volume ofchloroform was added to preserve the DNA from bacterial contaminationand to remove any residual proteins.

[0225] Plasmid and genomic DNA from B. campestris was digested withrestriction enzymes compatible with the multiple cloning site (MCS) ofpUC118 (Vieira and Messing, 1982). Restriction endonucleases fromBoehringer-Mannheim (Dorval, Quebec, Canada), Promega (Madison, Wis.;Burlington, Ontario, Canada), and New England Biolabs (Mississauga,Ontario, Canada) were used as recommended by the suppliers. DNAfragments were separated in either 0.65% 40 mM Tris-acetate/1 mM EDTA(TAE) or 0.7% 90 mM Tris-borate/2 mM EDTA (TBE) agarose gels run at8.5V/cm and blotted by the method of Southern (1975) onto Hybond N(Amersham Corp.) nylon membranes. Molecular weights of fragments weredetermined by multiple regression analysis based on mobility standardsof EcoRI—HindIII digests of bacteriophage lambda (Promega).

[0226] For colony blots, Hybond N membrane was placed on top of thecolonies, lifted off the plate, incubated for 6 to 8 h on a newLB-ampicillin plate, where necessary, to allow growth of the cells, andthe colonies were lysed on the membrane in situ.

[0227] A degenerate 23-mer oligonucleotide probe, (APO-1;5′-AAAGATATTGG(ATC)AAAGG(ATC)ATTGG-3′) (SEQ ID NO:52) based on residues8 to 15 of the amino acid sequence, was used to locate the brochocin-Cstructural gene (brcA) in both Southern and colony blot hybridizations.Oligonucleotides based upon derived nucleotide sequences weresynthesized as needed (Department of Biological Sciences, University ofAlberta, Edmonton, AB) on an Applied Biosystems 391 PCR Matesynthesizer, quantified, and used for hybridizations or as primers fornucleotide sequencing without further purification. DNA probes wereradioactively end-labelled with [γ³²P]ATP (Amersham) with T4polynucleotide kinase (PNK; Promega) or nonradioactively byrandom-primed labelling with digoxigenin-dUTP (Boehringer-Mannheim). Areaction volume of 10 mL of the labelled oligonucleotide mixture (6 mLdistilled water, 1 mL 10× PNK buffer, 1 mL [1 pmol] APO-1 probe, 1 mLPNK, 1 mL [γ³²P]ATP) was added for every 3 mL of hybridization solution.The mixture was purified through a Sephadex G50 column to removeunincorporated ATP or added directly to the hybridization solution.Hybridizations were done at 37° C. overnight in hybridization solutioncontaining 6× SSPE buffer, ₅× Denhardt's Reagent (Sambrook et al., 1989)and 0.5% (v/v) SDS. After hybridization, two washes were donesequentially (25° C. for 25 min, 39° C. for 15 min) in 2× SSPE buffer,0.1% SDS. Where necessary, probes were stripped off membranes by washingat 95° C. for 2 min in 0.5% SDS and rehybridized. Autoradiograms wereexposed 24 to 48 h before developing in a Fuji film processor.

[0228] Isolation of small-scale plasmid DNA from E. coli strains wasperformed by the lysis by boiling method and large-scale DNA preparationby alkaline lysis (Sambrook et al., 1989). Large-scale plasmid DNA waspurified by equilibrium centrifugation at 49 000 rpm (Ti 70.1 rotor) for20 h in a CsCl-ethidium bromide gradient and dialyzed in TE buffer.

[0229] Cloning of the brcA Gene.

[0230] Genomic DNA was digested to completion with EcoRI. Fragments of4.2 kb corresponding to the hybridization signal identified with APO-1were excised from the gel and placed in 6,000 to 8,000 molecular weightcut-off Spectrapor (Los Angeles, Calif.) dialysis tubing. The DNA waselectroeluted from the gel and into the tubing by electrophoresis at200V for 20 min in 0.5% TEE buffer. The DNA was purified by extractingonce with phenol/chloroform:isoamyl alcohol (24:1), once withchloroform:isoamyl alcohol, and precipitated with 2 volumes of 95%ethanol and one-tenth volume of 3 M sodium acetate (pH 5.2). Theresulting fragments were cloned into the EcoRI site of the MCS in pUC118using T4 DNA ligase (Promega) at 25° C. for 3 h in ligation bufferwithout polyethylene glycol and dithiothreitol. Colonies were screenedby a-complementation (Vieira and Messing, 1982). Colony blots were doneto discriminate the white colonies for the correct DNA insert.Small-scale plasmid isolations were done on presumptive positive clonesand the plasmids were digested with TaqI. The clones were grouped intoclasses based on similarities in their restriction patterns. Clones weredigested with EcoRI, blotted by the method of Southern (1975), andhybridized with APO-1 to confirm the presence of the brcA gene. Theplasmid identified to carry the correct 4.2 kb insert in pUC118 wasnamed pAP7.4. A smaller PstI fragment of 1.4 kb was further identifiedfrom this plasmid to hybridize to APO-1 and this was subcloned intopUC118 (Pap4.6).

[0231] Nucleotide Sequencing of Plasmid DNA:

[0232] The plasmid pAP4.6 served as the initial template DNA fornucleotide sequencing by Taq DyeDeoxy Cycle sequencing on an AppliedBiosystems 373A sequencer using the universal forward and reverseprimers of pUC118. Site-specific 18-mer primers based on newly sequencedDNA were synthesized for further sequencing. The recombinant plasmid,pAP7.4, was used as the template DNA in subsequent sequencing runsto-deduce the complete sequence of the structural gene (brcA), theregions flanking the structural gene, and for sequencing of thecomplementary strand.

[0233] Heterologous and Homologous Expression Studies of Brochocin-C.

[0234] The 4.2 kb insert in pAP7.4 was subcloned into the EcoRI site ofthe shuttle vector pGKV210 to create the recombinant plasmid pAP8.6.This plasmid was subsequently used to transform selected strains byelectroporation with a Gene-Pulser (Bio-Rad Laboratories Canada Ltd.,Mississauga, ON) at 25 mFD and 200 ohms resistance. TABLE 9 Inhibitoryspectrum of Brochothrix campestris ATCC 43754 determined by direct anddeferred antagonism on APT agar Indicator Direct Deferred Bacillusmacerans ATCC 7048 ƒƒ ƒƒ B. cereus ATCC 14579 ++ +++ Brochothrixcampestris ATCC 43754 ƒƒ ƒƒ B. thermosphacta B1 ++ ++++ B. thermosphactaB2 ++ ++++ B. thermosphacta B3 ++ ++++ B. thermosphacta B4 ++ ++++ B.thermosphacta B5 ++ ++++ B. thermosphacta B7 ++ ++++ B. thermosphacta B8++ ++++ B. thermosphacta B9 ++ ++++ B. thermosphacta B10 ++ ++++ B.thermosphacta B11 ++ ++++ B. thermosphacta B12 + ++++ B. thermosphactaB13 ++ ++++ B. thermosphacta B14 + ++++ B. thermosphacta B15 ++ ++++ B.thermosphacta B16 + ++++ B. thermosphacta L90 + ++++ B. thermosphactaNF4 ++ ++++ B. thermosphacta C420 + ++++ B. thermosphacta I41 ++ +++Carnobacterium piscicola LV17 ++++ ++++ C. piscicola LV17A ++++ ++++ C.piscicola LV17B ++++ ++++ C. piscicola LV17C ++++ ++++ C. piscicolaC2/8B ++++ ++++ C. piscicola C2/8A ++++ ++++ C. piscicola UAL26 +++ ++++C. piscicola UAL26/8A +++ ++++ C. piscicola UAL26/8B ++++ ++++ C.divergens LV13 +++ ++++ C. divergens 9/8A +++ ++++ C. divergens 9/8B +++++++ Clostridium bifermentans ATCC19299 +++ ++++ C. butyricum ATCC 8260ND +++ C. pasteurianum ATCC 6013 ND +++ Enterococcus faecalis ATCC 19433+++ ++++ E. faecalis ATCC 7080 +++ +++ E. faecium ATCC 19434 +++ ++++ E.durans ATCC 11576 +++ ++++ Lactobacillus sake Lb706 +++ ++++ L.plantarum ATCC 4008 ƒƒ ƒƒ Lactococcus lactis ATCC 11454 ƒƒ + L. lactisUAL 245 + + L. lactis UAL 276 ND + Leuconostoc gelidum UAL 187 ++ +++ L.gelidum UAL 187.13 + ++ L. gelidum UAL 187.22 ++ +++ L. mesenteroidesATCC 23386 ƒƒ ƒƒ L. mesenteroides Y105 ƒƒ ++ Listeria innocua ATCC 33090++ +++ L. monocytogenes Scott A +++ ++++ L. monocytogenes UAL 42 ++ +++L. monocytogenes ATCC 15313 + ++ Pediococcus acidilactici ATCC 8042 + ++Staphylococcus aureus S6 ++ ++ S. aureus S13 ++++ ++++

Example 7 Novel Bacteriocin Nucleotide and Amino Acid Sequences(Enterocin 900)

[0235]Enterococcus faecium 900 produces a chromosomally mediated broadspectrum bacteriocin. The forward operon is referred to as SEQ ID NO:28.The bacteriocin consists of 71 amino acids herein referred to as SEQ IDNO:30 and its nucleotide sequence is herein referred to as SEQ ID NO:29.This bacteriocin has activity against other strains of Enterococcusspecies as well as many other organisms as indicated in Tables 3 and A.

[0236] Purification of Enterocin 900.

[0237] For purification of the Enterococcus faecium BFE 900 bacteriocinthe culture was grown in 2.5 l APT broth for 18 h at 30° C. The culturewas was heated at 70° C. for 35 min to inactivate proteases andcentrifuged at 10 000 rpm for 40 min. The supernatant was termedfraction I. Fraction I (2.5 l) was loaded onto an amberlite XAD-8(Pharmacia) hydrophobic interaction chromatography column and the columnwas washed with 3 l of 0.05% trifluoroacetic acid (TFA), and 2 l of 20%ethanol (EtOH)+0.05% TFA. Bacteriocin was eluted with 2 l of 40% EtOH+0.05% TFA. The pH of the eluate was adjusted to pH 5.0 and the eluatewas reduced to 47 ml at 37° C. in a rotary evaporator under vacuum. Theresulting fraction (fraction II) was pH adjusted (pH 5.0) and loadedloaded onto a carboxymethyl-cellulose CM22 (Whatman Biochemicals,Maidstone, Kent, England) cation exchange column (34 cm, 1.3 cm I.D.)that was pre-equilibrated with 20 mM sodium acetate buffer pH 5.0 (SAB).The column was washed with 100 ml SAB and 60 ml volumes of SAB with 40,80, and 120 mM NaCl added. Bacteriocin was eluted with 60 ml SAB with200 mM NaCl added. The bacteriocin containing eluate was loaded onto aSep Pak C18 reverse phase column (Waters) which was pre-equilibratedaccording to manufacturers instructions. The column was washed with 20ml of distilled water and 10 ml of 40% ethanol. Bacteriocin was elutedwith 10 ml of 70% ethanol, frozen overnight at −80° C. and subsequentlyfreeze dried. The freeze dried protein was resuspended in 1.5 ml 0.05%TFA (fraction III) and purified using a Beckman System Gold HPLC. ForHPLC purification 100 μl aliquots were applied to a C₁₈ reverse phasecolumn (Waters Delta-Pak; 8×100 mm; 15 μm particle size; 300□ (30 nm)pore size; flow rate 1.0 ml/min; mobile phase,

[0238]0.05% TFA [A] and 95% ethanol in 0.05% TFA [B]). Bacteriocin waseluted by a gradient method (first 40% to 60% solvent B in 7 min andthen 60 to 70% solvent B in 10 min). Fractions were monitored for A₂₁₈and for activity against the indicator strain. The puritiy of thefraction was determined by tricine gel electrophoresis.

[0239] Bacteriocin activity of fractions I, II and III was determined bythe critical dilution method described in section 2.1.1, usingLactobacillus sake DSM 20017 as indicator organism. Proteinconcentration of these fractions was determined by the dye bindingmethod of Bradford (Bradford, 1976).

[0240] Protein Sequencing.

[0241] Protein sequencing was performed by Edman degradation on anautomated sequencer. To determine whether the structural enterocin geneindeed resides on the chromosome an oligonucleotide probe based on thefirst 11 amino acids of enterocin 900 was constructed and used to probechromosomal DNA. The probe CF01 consisted of the following 32nucleotides: GAA AAT GAT CAT (C/A) G (T/A) ATG CC (T/A) AAT GAA CT (T/A)AA and had a T_(M) of 82° C. Chromosomal DNA was isolated by the methodsof Quadri et al., 1994 and digested with the restriction enzymes EcoRI,PstI and HindIII before running on a 0.7% agarose gel. DNA wastransferred to hybond membrane by Southern blotting as described inSambrook et al. 1989. The probe CF01 was end labelled with ³²P-[γ-ATP]and hybridized to the DNA as described by Sambrook et al. 1989. Theprobe hybridized to a 2.2 kbp HindIII fragment and a 6.5 kbp EcoRI/PstIfragment.

[0242] The 2.2 kbp HindIII fragment was cloned into pUC118 contained inE. coli DH5α and sequenced. The nucleotide sequence analysis wasperformed by sequencing the DNA in both orientations by dideoxy-chainmethod of Sanger and associates (1977). DNA was sequenced by Tag DyeDeoxy Cycle sequencing on an Applied Biosystems DNA sequencer (AppliedBiosystem, Foster City, Calif.).

Example 8 A Food-Grade Plasmid pCD3.4

[0243] Large scale plasmid preparation from C. divergens LV13 was doneas described for C. piscicola LV17A (Worobo et al., 1994). Other DNAmanipulations were based on those described by Sambrook et al. (1989).Pfu DNA polymerase (Stratagene, LaJolla, Calif.), restrictionendonucleases and T4 DNA ligase were obtained from Promega (Madison,Wis.), Bethesda Research Laboratories (Burlington, ON), BoehringerMannheim (Dorval, PQ), New England Biolabs (Mississauga, ON) and usedaccording to the suppliers' recommended procedures. Step-wise deletionderivatives for sequencing were prepared using the Erase-a-Base® system(Promega) and DNA fragment recovery was done using Geneclean II® (Bio101 Inc., LaJolla, Calif.). Oligonucleotides prepared as sequencing andPCR primers were synthesized on an Applied Biosystems (model 391) PCRMate synthesizer. Double stranded DNA was sequenced by Taq DyeDeoxyCycle sequencing on an Applied Biosystems (model 373A) sequencer.

[0244] The nucleotide sequence is herein referred to as SEQ ID NO:14.From the nucleotide sequence and the restriction maps (FIG. 8) one ofordinary skill in the art can identify a variety of suitable locationsfor inserting other genes without undue experimentation. This plasmidcan be use to insert genes for use in probiotics, meat, milk products,food or food products. The bacteriocin Divergicin A was derived fromthis plasmid (Worobo et al. 1995) and the signal peptide nucleotidesequence is used in other sections of this application is referred to asSEQ ID NO:19 and the corresponding amino acid sequence is SEQ ID NO:20.

Example 9 Methods for Testing Organisms for Preservation of Meat andOrganisms that will Preserve Meat.

[0245] Bacterial Cultures and Identification of Lb. sake 1218. Thelactic acid bacteria used in this study are listed in Table 1. Lb. sake1218 is a sulfide-producing LAB isolated from modified atmospherepackaged pork stored at −1° C. (McMullen and Stiles, 1993). The strainwas initially identified by McMullen and Stiles (1993) using standardtechniques (Montel et al., 1991; Schillinger and Lucke, 1987), and itsidentity was confirmed in this study with the following biochemical andcultural tests: production of slime from sucrose; ability to grow onacetate agar (Cavett, 1963); reduction of tetrazolium (Wilkinson andJones, 1977); final pH in La-broth (Reuter, 1970; Shaw and Harding,1984); presence or absence of meso-diaminopimelic acid (Kandler andWeiss, 1986); sugar-fermentation pattern (Shaw and Harding, 1985); andlactic acid isomer determination by an enzymatic-UV method (BoehringerMannheim, 1987). Lb. sake 1218 was tested for bacteriocinogenic activityagainst all of the Leuc. gelidum strains by direct and deferredinhibition tests (Ahn and Stiles, 1990a; Ahn and Stiles, 1990b).

[0246] Inhibition of Lb. sake 1218 by Leuc. gelidum Strains in APTBroth.

[0247] Growth rates of Leuc. gelidum UAL187 and its variants weredetermined in pure culture at 2 and 25° C. in APT broth (DifcoLaboratories Inc., Detroit, Mich.) containing 2% glucose, or in modifiedAPT broth (mAPT) made according to Difco (Difco Manual, 1984) butcontaining 0.05 or 0.1% glucose inoculated at 4.2 to 4.3 log CFU/ml.Initial pH of APT broth was adjusted to 5.6 or 6.5. Competitive growthstudies of Leuc. gelidum UAL187, UAL187-22 or UAL187-13 with Lb. sake1218 were done in MAPT containing 0.1% glucose and initial pH adjustedto 5.6.

[0248] Inocula for all experiments were grown in APT broth at 25° C. for18 h. Cells were washed three times by centrifugation at 16,000× g,washed with sterile, 0.1% peptone water and resuspended in peptone waterat the desired cell density. Samples for bacterial enumeration werediluted in 0.1% peptone water and surface streaked onto MS agar,consisting of tryptone (10 g/l), yeast extract (5 g/l), fructose (2.5g/l), KH₂PO₄ (2.5 g/l), L-cysteine HCl (0.5 g/l), MgSO₄.7H₂O (0.2 g/l),MnSO₄.H₂O (0.05 g/l), calcium pantothenate (0.01 g/l), agar (20 g/l),Tween 80 (1 ml/l), and bromocresol green (0.1 g in 30 ml of 0.01 N NaOH)(20 ml/l) (Züñiga et al., 1993). This medium differentiated theheterofermentative Leuc. gelidum colonies (white color) fromhomofermentative Lb. sake 1218 colonies (blue color). Representativecolonies were checked by their phenotypic characteristics to determinethe reliability of the differentiation (see below). MRS (Difco)-sorbicacid agar (Anon, 1987) was used for selective enumeration of Lb. sake1218. Plates were incubated at 25° C. for 3 days. pH was determined inall samples. Antimicrobial activity of leucocin A in the supernatant wasassayed by the spot-on-lawn method (Ahn and Stiles, 1990a; Ahn andStiles, 1990b) with Carnobacterium divergens LV13 as the indicatorstrain. All experiments were done in duplicate.

[0249] Inoculation of Beef Samples.

[0250] Sterile, lean slices of beef (surface area 20 cm²) were excisedaseptically from normal pH longissimus dorsi muscle as described byGreer and Jones, 1991. Beef slices were suspended from sterile clips andimmersed for 15 sec in a bacterial suspension containing 10⁵ CFU/ml forLeuc. gelidum and 10³ CFU/ml for Lb. sake, and allowed to air dry at 25°C. for 15 min. This gave an inoculum of approximately 10⁴ CFU cm⁻² forLeuc. gelidum and 10² CFU cm⁻² for Lb. sake. An equal number of beefslices was immersed in sterile, 0.1% peptone water for use as controls.

[0251] Beef Storage.

[0252] Three inoculated beef slices from each sample were placed insterile Stomacher bags (Seward Medical, U.K.), enclosed in gasimpermeable foil laminate bags (Printpac-UEB, Auckland, New Zealand) andvacuum packaged using a Captron III Packaging System (RMF, Grandview,Mo.). Vacuum packaged beef samples were stored at 2° C. for 8 weeks andsamples were removed for analysis after 0, 1, 2, 3, 4, 4.5, 5, 6 and 8weeks of storage. Three or four independent trials were done formicrobiological content and sensory analysis of each combination ofbacterial inocula, except for meat inoculated with pure cultures ofLeuc. gelidum UAL187, UAL187-22 and UAL187-13, for which only one trialwas done.

[0253] Bacterial Sampling and Determination of Antimicrobial Activity onMeat.

[0254] At each sampling time, three beef slices from one package werehomogenized separately in a Colworth Stomacher 400 (Baxter DiagnosticsCorp., Canlab Division, Edmonton, AB Canada) in 90 ml of sterile 0.1%peptone water. Samples were diluted and surface plated onto M5 orMRS-sorbic acid agar and incubated at 25° C. for 3 days. The reliabilityof detection of the Leuconostoc strain was checked by the ability toproduce slime on APT agar containing 2% sucrose. An average of 8colonies of each of the Leuc. gelidum variants was picked from M5 agarplates from meat samples analyzed after 3 or 8 weeks of storage. Thesecolonies were grown in APT broth, and examined for purity bycarbohydrate fermentation patterns (Shaw and Harding, 1985), some werealso examined for plasmid profiles (Ahn and Stiles, 1990b) and forbacteriocin production by overlayering with the indicator strain. Afterenumeration, M5 plates were overlayered with soft APT agar (0.75% agar)containing 1% of an overnight culture of C. divergens LV13 or Lb. sake1218 to determine antimicrobial activity by the deferred inhibitiontest.

[0255] Production of leucocin A during growth of the producer strain onbeef was determined by a modification of the procedure described byRuiz-Barba et al. (Ruiz-Barba et al., 1994). One beef slice washomogenized in 90 ml of 0.1% peptone water, heated in a boiling waterbath for 15 min, cooled rapidly on ice and centrifuged at 8,000× g for15 min. Ammonium sulfate (Fisher Scientific; Fair Lawn, NJ) was added to70% saturation, stirred at 4° C. overnight and centrifuged at 20,000× gfor 1 h at 0.5° C. The precipitate was resuspended in 1.5 ml of sodiumphosphate buffer (50 mM, pH 7.0) and activity was determined by thespot-on-lawn method (Ahn and Stiles, 1990a; Ahn and Stiles, 1990b) usingC. divergens LV13 as indicator. The presence of bacteriocin wasconfirmed by adding 10 μl of Pronase E (1 mg/ml; Sigma Chemical Co., St.Louis, Mo.) to appropriate samples of supernatant.

[0256] Sensitivity of Lb. sake 1218 to leucocin A.

[0257] After 8 weeks of storage under vacuum at 2° C., one of the beefslices from each inoculum type was homogenized in 90 ml of sterile, 0.1%peptone water. From each sample, 75 μl of liquid was withdrawn and mixedwith 7.5 ml of “soft” MRS-sorbic acid agar (0.75% agar) and plated onMRS-sorbic acid agar (1.5% agar) for selective growth of Lb. sake 1218.Supernatants of APT broth cultures of Leuc. gelidum UAL187 or UAL187-13grown at 25° C. for 18 h were adjusted to pH 6.5 with 1 N NaOH andheated at 65° C. for 30 min. From these preparations, 20 μl ofappropriate two-fold dilutions was spotted onto the Lb. sake 1218indicator lawns to be tested for sensitivity to leucocin A. Plates wereincubated anaerobically at 25° C. overnight and observed for zones ofinhibition.

[0258] Sensory Assessment of Beef Samples.

[0259] Qualitative analysis of odor acceptability, based on detection ofsulfur odors in vacuum packed beef samples, was done as described byMcMullen and Stiles, 1994. An experienced five-member panel was used.Each packaged sample containing three slices of beef was filled with 200ml helium, and 5 ml of headspace gas was withdrawn for sensory analysisthrough a “sticky nickel” (Mocon Modern Controls Inc., Minneapolis,Minn.) sampling port with a gas tight syringe (SGE, Mandel Scientific,Guelph, Ontario) equipped with a button lock. Acceptability was judgedby absence or presence of sulfur odor. A sample was deemed spoiled if50% or more of the panelists rejected the sample because of a sulfurodor.

[0260] Characterization and Identification of Lb. sake 1218.

[0261] The Gram-positive, rod-shaped, catalase- and oxidase-negativestrain 1218 was classified as Lb. sake based on its followingcharacteristics: no gas from glucose; growth on acetate agar;degradation of arginine; unable to reduce tetrazolium; absence ofmeso-diaminopimelic acid in the cell wall; production of D- and L-lacticacid isomers; final pH<4.15 in La-broth; and the following carbohydratefermentation pattern: amygdalin (−), arabinose (+), cellobiose (−),fructose (+), glucose (+), inulin (−), inositol (−), lactose (−),maltose (−) mannitol (−), mannose (+), melezitose (−), melibiose (+),raffinose (−), ribose (+), salicin (−) and sucrose (+). No acids wereproduced from glycerol or pyruvate. The organism grew in the presence of6.5% NaCl but not at 45° C. Preliminary experiments showed that Lb. sake1218 produced strong sulfurous off odors when inoculated onto vacuumpackaged beef, but not on beef stored under aerobic conditions. Lb. sake1218 was not found to be bacteriocinogenic against any of the Leuc.gelidum variants when tested by deferred and spot-on-lawn techniques. MSagar did not give a reliable differentiation between the test strains.More reliable information was obtained from the counts on MRS-sorbicacid agar to enumerate Lb. sake 1218.

[0262] Inhibition of Lb. sake 1218 by Leuc. aelidum Strains in APTbroth.

[0263] At 25° C. the three isogenic variants of Leuc. gelidum UAL187 hadidentical doubling times of 3.85 h when grown as pure cultures or incombination with Lb. sake 1218. In MAPT with initial glucoseconcentrations of 0.05, 0.1 or 2% or initial pH values of 5.6 or 6.5 ofthe growth medium did not affect the growth rate of the Leuc. gelidumvariants. At 2° C. the initial doubling times for Leuc. gelidum UAL187,UAL187-13 and UAL187-22 were similar, averaging 1.75 days; but afterfour to eight days of incubation the doubling time of Leuc. gelidumUAL187-22 increased to 3.15 days. This change in growth rate could notbe attributed to glucose concentration or pH of the growth medium orwhether Leuc. gelidum UAL187-22 was grown as pure culture or togetherwith Lb. sake 1218.

[0264]Lb. sake 1218 grown in APT broth in mixed culture with Leuc.gelidum UAL187 or UAL187-22 at 25° C. was inhibited at the time (17 h)that antimicrobial activity was detected in the supernatant (FIG. 9).Growth of Lb. sake 1218 resumed after 21 h, coinciding with a decreasein antimicrobial activity, and reached a population of approximately 10⁷CFU/ml after extended incubation of 100 h at 25° C. (FIG. 9). Lb. sake1218 grew rapidly in pure culture or in mixed culture with Leuc. gelidumUAL187-13 (FIG. 9). Antimicrobial activity was not detected in thesecultures.

[0265] Growth of Lb. sake 1218 in APT broth at 2° C. was inhibited inmixed culture with Leuc. gelidum UAL187 after 8 d of incubation,coinciding with the time that antimicrobial activity was first detectedin the supernatant (FIG. 10). The cell density of Lb. sake 1218decreased to the minimum detection limit after 12 d of incubation, butgrowth resumed after approximately 30 to 35 d of storage (FIG. 10). Lb.sake 1218 grew rapidly at 2° C. in pure culture and in mixed culturewith Leuc. gelidum UAL187-13 (FIG. 10). Antimicrobial activity was notdetected in these cultures. Lb. sake 1218 in mixed culture with Leuc.gelidum UAL187-22 grew actively for the first 15 d of incubation; afterwhich a rapid decline in cell counts of Lb. sake 1218 was observed,coinciding with the detection of antimicrobial activity (FIG. 10). After22 days of incubation there was a loss of antimicrobial activity and Lb.sake 1218 resumed its growth. pH did not change more than 0.2 units fromthe initial value in any of the experiments done with MAPT.

[0266] Growth of Bacteria and Detection of Bacteriocin on VacuumPackaged Beef.

[0267] The data shown in FIG. 11 illustrate the growth of the threeisogenic strains of Leuc. gelidum UAL187 inoculated as pure cultures orco-inoculated with Lb. sake 1218 on beef stored under vacuum at 2° C.Leuc. gelidum UAL187 and UAL187-13 again exhibited identical growthrates, while Leuc. gelidum UAL187-22 grew at a considerably slower rate.Growth and survival of Lb. sake 1218 alone or in mixed culture with theisogenic variants of Leuc. gelidum is shown in FIG. 12. Lb. sake 1218grew rapidly as a pure culture on vacuum packaged beef producing asulfurous odor within three weeks at 2° C. Pronounced inhibition of Lb.sake 1218 was observed in three out of four trials in which Lb. sake1218 was co-inoculated with Leuc. gelidum UAL187 on meat. There was adelay of growth for 5 weeks with a 4 log lower count of Lb. sake 1218after 8 weeks of incubation. In a fourth trial, there was a delay of twoweeks before initiation of growth of Lb. sake 1218 and relatively lowmaximum count of 105 to 106 log CFU cm⁻2 was observed. These data werenot included in the means calculated for FIG. 12. Similar growth of Lb.sake 1218 but with approximately one log lower maximum count than inpure culture was observed when Lb. sake 1218 was co-inoculated withLeuc. gelidum UAL187-13. A slight delay in initiation of growth and areduction of 0.5 to 1 log units in maximum count was observed when Lb.sake 1218 was co-inoculated with Leuc. gelidum UAL187-22. Comparisonwith pure culture studies indicated that growth of Leuc. gelidum UAL187and its isogenic variants was not affected by the presence of Lb. sake1218 in any trial. The identity of each variant of Leuc. gelidum wasconfirmed by comparison of plasmid profiles, carbohydrate fermentationpatterns and slime production of colonies isolated after eight weeks ofstorage from each experiment.

[0268] The possibility that Lb. sake 1218 developed resistance toleucocin A during the trial with extended growth in the presence ofLeuc. gelidum UAL187 was tested. Spot-on-lawn tests of isolates of Lb.sake 1218 were done after 8 weeks of storage. Results showed that Lb.sake 1218 was sensitive to 800 AU ml⁻1 in heat treated supernatant ofLeuc. gelidum UAL187 grown in APT. The same sensitivity was observed forisolates of Lb. sake 1218 grown as pure cultures or in mixed culturewith Leuc. gelidum UAL187-22 or UAL187-13. Growth of Lb. sake 1218 withextended incubation was apparently due to loss of activity of leucocin Arather than development of resistant strains of Lb. sake 1218.

[0269] Antimicrobial activity that was sensitive to pronase E wasdemonstrated for extracts prepared from beef samples co-inoculated withLeuc. gelidum UAL187 and Lb. sake 1218. The antibacterial activity onthe meat persisted from two up to eight weeks of storage, but the levelof activity was near the lowest detectable limit and activity could notbe detected on all samples that were tested. At least half of the trialswere positive at each sampling time. Antimicrobial activity was alsoobserved on beef co-inoculated with Leuc. gelidum UAL187-22 and Lb. sake1218 after six weeks of storage. No activity was observed for beefco-inoculated with Leuc. gelidum UAL187-13 and Lb. sake 1218. Leuc.gelidum UAL187 and UAL187-22 retained their bacteriocinogenic potentialat all storage intervals when tested for antagonistic activity by thedeferred inhibition test.

[0270] Detection of Spoilage of Beef Samples.

[0271]Leuc. gelidum UAL187 completely inhibited sulfur-mediated spoilageof beef by Lb. sake 1218 for up to eight weeks, except in two of fourtrials, where spoilage was detected in samples taken at 4.5 weeks butnot at 6 and 8 weeks of storage at 2° C. Spoilage produced by Lb. sake1218 in the presence or absence of Leuc. gelidum UAL187-22 or UAL187-13was detected within 3 to 4.5 weeks of storage and illustrated by arrowsin FIG. 12. No spoilage was detected in beef samples inoculated withpure cultures of Leuc. gelidum UAL187, UAL187-22 or UAL187-13 and storedfor up to eight weeks under vacuum at 2° C.

[0272] Preservation of Pork.

[0273] Application of modified atmosphere packaging for retail marketingof pork cuts was studied. Experiments were designed to determine: (1)effects of storage conditions on keeping quality and the prevailingmicroflora on the meat cuts; (2) the potential to access distant marketswith retail-ready cuts using this technology; and (3) the effect ofinoculation of retail cuts with selected lactic acid bacteria (LAB) onkeeping quality and the use of headspace gas analysis to monitorspoilage.

[0274] To examine the effects of storage conditions pork loin cutsprepared with two levels of initial bacterial load were packaged inthree films of different gas transmission in an atmosphere containing40% CO₂/60% N₂ and stored at −1, 4.4 and 10° C. Temperature was theoverriding factor influencing storage life. Spoilage at each storagetemperature could be attributed to the growth of different groups ofbacteria and was influenced by package type. Storage life of pork cutsin packages with low oxygen transmission rates was 5 or 8 weeks at 4.4or −1° C., respectively. Listeriae were detected as part of theprevalent microflora on samples stored at −1° C., but not on samplesstored at 4.4 or 10° C. A total of 162 (30%) of LAB isolated from themeat samples produced inhibitory substances against a range of indicatorstrains.

[0275] Samples for studies to simulate storage conditions to accessdistant markets with retail-ready cuts of pork were packaged in 100% CO₂in plastic film with extremely low gas transmission and stored at −1.5°C. for three weeks. Reference samples were held at −1.5° C. for theduration of the study. After transfer of samples to 4 and 7° C., samplesremained acceptable for retail sale for 2 and 1 weeks, respectively.Appearance of the cuts was the main factor limiting storage life;however, confinement odor became a potential problem for consumeracceptance of the product with extended storage.

[0276] Studies of inoculated retail-ready cuts of pork packaged in 100%CO₂ and stored at 4° C. revealed that the type of bacteriocinogenic LABaffected the storage life of the meat. Sulphur odors were detected onmeats inoculated with Carnobacterium piscicola LV17 or Leuconostocgelidum UAL187 but not with Lactobacillus sake Lb706 using methodsdescribed for beef. Detection of sulfur compounds in the headspace gasat the time that the sensory panel detected off-odors, indicated thatmonitoring of these compounds is an objective measure of spoilage.

[0277] The studies demonstrated that there is good potential to applymodified atmosphere packaging technology to retail cuts of pork. Withadequate temperature control, storage life can be extended for weeksbeyond what is possible with aerobic packaging.

[0278] Assessment of the spoilage potential of selected strains of LABis imperative before they can be exploited as biopreservatives forachieving a predictable storage life of retail-ready products.

Example 10 (Method for Using Bacterocins for Treating Infections)

[0279] For the treatment of animal or human diseases, purified orpartially purified bacteriocins are used for topical application orinternal use.

[0280] The bacterocins are purified or partially purified by a varietyof methods including, without limitation, the methods described hereinor those described by Henderson and associates (1992); Hechard andassociates (1992); Hasting and associates (1991); Quadri and associates(1993) or Worobo and associates (1994) or may be able to be obtainedcommercially obtained commercially (Quest; Flavors & Food IngredientsCo., Rochester, N.Y.).

[0281] The formulations for delivery are similar to other bacterocins,and one of ordinary skill in the art can determine which formulation touse without undue experimentation. The concentration of bacterocinrequired for one of these formulations can be determined by comparingunits/pg of a known bacterocin to the novel bacteriocin. Theconcentration of the novel bacteriocin should be set so that theconcentration of the novel bacteriocin active units/ml is 0.1 to 10times the activity of the control.

Example 11 Use of Organisms Containing Bacteriocin Genes to PreserveFood

[0282] To prevent food poisoning, milk products lactobacteriumcontaining a Gram-negative bacteriocin (i.e. Colicin V) (these organismscould also contain other bacteriocins) can be added to the product. Foryogurt, 10⁸ to 10⁹ lactobacillus bacteria are added to milk. To improvethe shelf life of this product (0.01% to 100 % of these organisms addedcould contain the desired plasmid). This same method can be used forprotection of cheese but the host bacterium and number of organismsincoluated into the milk is dependent on the type of cheese, one ofordinary skill in the art can determine what type of organism to use.

Example 12 Treatment of Infections or Bacteria Disorders

[0283] For intestinal infections such as food poisoning due toparticular organism (E.coli; Salmonella, etc.), an anti-diarrheatreatment contains 10⁶ to 10⁸ harmless organisms (i.e. lactobacillusstrains) in a buffered solution, suitable to be administered orally. Theorganisms contain a bacteriocin, in a food-grade plasmid, that inhibitthe growth of the common diarrhea-causing organisms (i.e. bacteriocinsactive against gram-negative organisms—Colicin V). These same organismsare also added to a buffered ointment suitable for vaginaladministration.

REFERENCES

[0284] Ahn, C., and M. E. Stiles. 1990. Plasmid-associated bacteriocinproduction by a strain of Carnobacterium piscicola from meat. Appl.Environ. Microbiol. 56:2503-2510.

[0285] Allison, G. E., Worobo, R. W., Stiles, M. E., and Klaenhammer, T.R. (1995b) Heterologous expression of the lactacin F peptides byCarnobacterium piscicola LV17 Appl Environ Microbiol. 61: 1371-1377.

[0286] Anon. 1987. de Man, Rogosa and Sharpe agar with sorbic acid(MRS-S agar). Int. J. Food Microbiol. 5:230-232.

[0287] Axelsson, L., A. Holck, S.-E. Birkland. T. Aukrust. and H. Blom.1993. Cloning and nucleotide sequence of a gene from Lactobacillus sakeLb706 necessary for sakacin A production and ammunity. Appl. Environ.Microbiol 59:2868-2875.

[0288] Axelsson, L., and A. Holck. 1995. The genes involved inproduction of and immunity to sakacin A, a bacteriocin fromLactobacillus sake Lb706. J. Bacteriol. 177:2125-2137.

[0289] Barefoot, S. F., and T. R. Klaenhammer (1983). Detection andactivity of lactacin B, a bacteriocin produced by Lactobacillusacidophilus. Appl. Environ. Microbiol. 45:1808-1815.

[0290] Birnboim, H. C., and Doly, J. (1979) A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. Nucleic Acids Res7:1513-1523.

[0291] Blight, M. A., and I. B. Holland. (1990) Structure and functionof haemolysin B, P-glycoprotein and other members of a novel family ofmembrane translocators. Mol Microbiol 4: 873-880.

[0292] Boehringer Mannheim. 1987. D(−)-Lactate dehydrogenase (D-LDH).Biochemica information, p. 45. Boehringer Mannheim Biochemicals,Indianapolis, Ind.

[0293] Borch, E. and Agerhem, H. (1992) Chemical, microbial and sensorychanges during the anaerobic cold storage of beef inoculated with ahomofermentative Lactobacillus sp. or a Leuconostoc sp. Int. J. FoodMicrobiol. 15, 99-108.

[0294] Borchert, T. V., and V. Nagarajan. 1991. Effect of signalsequence alterations on export of levansucrase in Bacillus subtilis. J.Bacteriol. 173:276-282.

[0295] Bukhtiyarova, M., Rongguang, Y., and Ray, B. (1994) Analysis ofthe pediocin AcH gene cluster from plasmid pSMB74 and its expression ina pediocin-negative Pediococcus acidilactici strain. Appl EnvironMicrobiol 60: 3405-3408.

[0296] Bureau Central de la CIE, 4, Avenue du Recteur Poincare, 75782Paris Cedex 16, France.

[0297] Cao, G., A. Huhn, and R. E. Dalbey. 1995. The translocation ofnegatively charged residues across the membrane is driven by theelectrochemical potential: evidence for an electrophoresis-like membranetransfer mechanism. EMBO J. 14:866-875.

[0298] Casadaban, M. C., and S. N. Cohen. 1980. Analysis of gene controlsignals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol.138:179-207.

[0299] Casadaban, M. J. (1976) Transposition and fusion of the lac genesto selected promotors in Escherichia coli using bacteriophage lambda andMu. J Mol Biol 104: 541-555.

[0300] Cavett, J. J. 1963. A diagnostic key for identifying the lacticacid bacteria of vacuum packed bacon. J. Appl. Bacteriol. 26: a53-470.

[0301] Chehade, H., and Braun, V. (1988) Iron-regulated synthesis anduptake of colicin V. FEMS Microbiol 52: 177-182.

[0302] Chopin, A., Chopin, M.-C., Moillo-Batt, A., and Langella, P.(1984) Two plasmid-determined restriction and modification systems inStreptococcus lactis. Plasmid 11: 260-263.

[0303] CIE (1976) Commission Internationale de l'Eclairage. 18thSession, London, England. September 1975. CIE Publication 36.

[0304] Collins, M. D., Rodrigues, U., Ash, C., Aguirre, M., Farrow, J.A. E., Martinez-Murcia, A., Philiips, B. A., Williams, A. M. andWallbanks, S. (1991) Phylogenetic analysis of the genus Lactobaci/lusand related lactic acid bacteria as determined by reverse transcriptasesequencing of 16S rRNA. FEMS Microbiol. Lett. 77, 5-12.

[0305] Dainty, R. H. and Mackey, B. M. (1992) The relationship betweenthe phenotypic properties of bacteria from chill-stored meat andspoilage processes. J. Appl. Bacteriol. Symp. Suppi. 73, 103S-114S.

[0306] Diep, D. B., L. S. HEvarstein, J. Nissen-Meyer, and I. F. Nes.1994. The gene encoding plantaricin A, a bacteriocin from Lactobacillusplantarum C11, is located on the same transcription unit as an agr-likeregulatory system. Appl. Environ. Microbiol. 60:160-166. Difco Manual.1984. 10th ed., p. 1071. Difco Laboratories Inc., Detroit, Mich.

[0307] Dinh, D., I. T. Paulsen, and M. H. Saier, Jr. 1994. A family ofextracytoplasmic proteins that allow transport of large molecules acrossthe outer membranes of Gram-negative bacteria. J. Bacteriol.176:3825-3831.

[0308] Edwards, R. A., Dainty, R. H. and Hibbard, C. M. (1985)Putrescine and cadaverine formation in vacuum packed beef. J. Appl.Bacteriol. 58, 13-19.Egan, A F. (1983) Lactic acid bacteria of meat andmeat products. Antonie van Leeuwenhoek 49,

[0309] Egan, A. F., Ford, A. L. and Shay, B. J. (1980) A comparison oflMicrobacterium thermosphacta and lactobacilli as spoilage organisms ofvacuum-packaged sliced luncheon meats. J. Food Sci. 1745-1748.

[0310] Fath, F. J., and R. Kolter. 1993. ABC transporters: bacterialexporters. Microbial. Rev. 57:995-1017.

[0311] Franke, C. M., Leenhouts, K. J., Haandrikman, A. J., Kok, J.,Venema, G., and Venema, K. (1996) Topology of LcnD, a protein implicatedin the transport of bacteriocins from Lactococcus lactis. J Bacteriol178: 1766-1769.

[0312] Fremaux, C., Ahn, C., and Klaenhammer, T. R. (1993) Molecularanalysis of the lactacin F operon. Appl Environ Microbiol 59: 3906-3915.

[0313] Fremaux, C., and T. R. Klaenhammer. 1994. Helveticin J, a largeheat-labile bacteriocin from Lactobacillus helveticus, p. 397-418. In L.De Vuyst and E. J.

[0314] Gasson, M. J. 1983. Plasmid complements of Streptococcus lactisNCDO 712 and other lactic streptococci after proptoplast-induced curing.J.Bacteriol. 154:1-9.

[0315] Gilson, L., H. K. Mahanty, and R. Kolter. 1987. Four plasmidgenes are required for colicin V synthesis, export, and immunity. J.Bacteriol. 169:2466-2470.

[0316] Gilson, L., H. K. Mahanty, and R. Kolter. 1990. Genetic analysisof an MDR-like export system: the secretion of colicin V. EMBO J.9:3875-3884.

[0317] González, B., P. Arca, B. Mayo, and J. E. Suárez. 1994.Detection, purification, and partial characterization of plantaricin C,a bacteriocin produced by a Lactobacillus plantarum strain of dairyorigin. Appl. Environ. Microbiol. 60:2158-2163.

[0318] Graham, D. C., and McKay, L. L. (1985) Plasmid DNA in strains ofPediococcus cerevisiae and Pediococcus pentosaceus. Appl EnvironMicrobiol 50: 532-534.

[0319] Greer, G. G., and S. D. M. Jones. 1991. Effects of lactic acidand vacuum packaging on beef processed in a research abattoir. Can.Inst. Food Sci. Technol. J. 24:161-168.

[0320] Greer, G. G. and Murray, A. C. (1991) Freezing effects onquality, bacteriology and retail case life of pork. J. Food Sci. 56,891-894.

[0321] Greer, G. G., Dilts, B. D. and Jeremiah, L. E. (1993)Bacteriology and retail case life of pork after storage in carbondioxide. J. Food Prot. 56, 689-693.

[0322] Héchard, Y. B. Dérijard, F. Letellier, Y. Cenatiempo. 1992Characterization and purification of mesentericin Y105, an anti-Listeriabacteriocin from Leuconostoc mesenteroides. Journal of GeneralMicrobiology. 138:2725-2731.

[0323] Hanna. M. O., Savell, J. W., Smith. G. C., Purser, D. E.,Gardner. F. A. and Vanderzant, C. (1983) Effcct of growth of individualmeat bacteria on pH. color and odor of aseptically preparedvacuum-packaged round steaks. J. Food Prot. 42. 216-221.

[0324] Hastings, J. W., M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas,and M. E. Stiles. 1991. Characterization of leucocin A-UAL 187 andcloning of the bacteriocin gene from Leuconostoc gelidum. J. Bacteriol.173:7491-7500.

[0325] Hastings, J. W. and Stiles, M. E. (1991) Antibiosis ofLeuconostoc gelidum isolated from meat. J. Appl. Bacteriol. 70, 127-134.

[0326] Hastings, J. W., Sailer, M., Johnson, K., Roy, K. L., Vederas, J.C., and Stiles, M. E. (1991) Characterization of leucocin A-UAL 187 andcloning of the bacteriocin gene from Leuconostoc gelidum. J Bacteriol173: 7491-7500.

[0327] Havarstein, L. S., D. B. Diep, and I. F. Nes. 1995. A family ofbacteriocin ABC transporters carry out proteolytic processing of theirsubstrates concomitant with export. Mol. Microbiol. 16:229-240.

[0328] Håvarstein, L. S., H. Holo, and I. F. Nes. 1994. The leaderpeptide of colicin V shares consensus sequences with leader peptidesthat are common among peptide bacteriocins produced by Gram-positivebacteria. Microbiology 140:2383-2389.

[0329] Henderson, J. T., A. L. Chopko, and P. D. van Wassenaar. 1992.Purification and primary structure of pediocin PA-1 produced byPediococcus acidilactici PAC-1.0. Arch. Biochem. Biophys. 295:5-12.

[0330] Higgins, C. F. (1992) ABC transporters: from microorganisms toman. Annu Rev Cell Biol 8: 67-113.

[0331] Holo, H, Nilssen, O. and Nes, I. F. (1991) Lactococcin A, a newbacteriocin from Lactococcus lactis subsp. cremoris: isolation andcharacterization of the protein and its gene. J Bacteriol 173:3879-3887.

[0332] Holo, H., and I. F. Nes. 1989. High-frequency transformation, byelectroporation fo Lactococcus lactis subsp. cremoris grown with glycinein osmotically stabilized media. Appl. Environ. Microbiol. 55:3119-3123.

[0333] Hui, F. M., and D. A. Morrison. 1991. Genetic transformation inStreptococcus pneumoniae: nucleotide sequence analysis shows comA, agene required for competence induction, to be a member of the bacterialATP-dependent transport family. J. Bacteriol. 173:372-381.

[0334] Hui, F. M., L. Zhou, and D. A. Morrison. 1995. Competence forgenetic transformation in Streptococcus pneumoniae: organization of aregulatory locus with homology to two lactococcin A secretion genes.Gene 153:25-31.

[0335] Izard, J. W., and D. A. Kendall. 1994. Signal peptides:exquisitely designed transport promoters. Mol. Microbiol. 13:765-773.

[0336] Jarchau, T., Chakraborty, T., Garcia, F., and Goebel, W. (1994)Selection for transport competence of C-terminal polypeptides derivedfrom Escherichia coli hemolysin: the shortest peptide capable ofautonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62amino acids of HlyA. Mol Gen Genet 245: 53-60.

[0337] Joerger, M. C., and T. R. Klaenhammer. 1990. Cloning, expression,and nucleotide sequence of the Lactobacillus helveticus 481 geneencoding the bacteriocin helveticin J. J. Bacteriol. 172:6339-6347.

[0338] Jung, G. (1991) in Nisin and Novel Lantibiotics. Jung, G., andSahl, H.-G. (eds). Leiden:Escom, pp1-34.

[0339] Kandler, O., and O. Weiss. 1986. Regular, nonsporeformingGram-positive rods, p. 1208-1260. In: P. H. A. Sneath, M. E. Mair, N.S., Sharpe, and J. G. Holt (ed.), Bergey's manual of systematicbacteriology, vol. 2. Williams and Wilkins, Baltimore, Md.

[0340] Klaenhammer, T. R. 1993. Genetics of bacteriocins produced bylactic acid bacteria. FEMS Microbiol. Rev. 12:39-85.

[0341] Kuipers, O. P., H. S. Rollema, W. M. de Vos, and J. Siezen. 1993.Biosynthesis and secretion of a precursor of nisin Z by Lactococcuslactis, directed by the leader peptide of the homologous lantibioticsubtilin from Bacillus subtilis. FEBS Lett. 330:23-27.

[0342] Laemmli, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London) 227:680-685.

[0343] Lee, C., P. Li, H. Inouye, E. R. Brickman, and J. Beckwith. 1989.Genetic studies on the inability of β-galactosidase to be translocatedacross the Escherichia coli cytoplasmic membrane. J. Bacteriol.171:4609-4616.

[0344] Leer, R. J., J. M. van der Vossen, M. van Giezen, J. M. vanNoort, and P. H. Pouwels. 1995. Genetic analysis of acidocin B, a novelbacteriocin produced by Lactobacillus acidophilus. Microbiology141:1629-1635.

[0345] Leisner, J., G Greer, B. Dilts, M. Stiles. 1994. Effect of growthof selected lactic acid bacteria on storage life of beef stored undervacuum and in air.

[0346] Leisner, J., G. Greer, and M. Stiles. 1996. Control of beefspoilage by a sulfide-producing lactobacillus sake strain withbacteriocinogenic leuconostoc gelidum UAL187 during anaerobic storage at2° C.

[0347] Lewus, C. B., Kaiser, A. and Montville. T. (1991) Inhibition offood-borne bacterial pathogens by bacteriocins from lactic acid bacteriaisolated from meat. Appl. Environ., Uicrobiol. 57. 1683-1688.

[0348] Li, P., J. Beckwith, and H. Inouye. 1988. Alteration of the aminoterminus of the mature sequence of a periplasmic protein can severelyaffect protein export in Escherichia coli. Proc. Natl. Acad. USA85:7685-7689.

[0349] Mandel, M., and A. Higa. 1970. Calcium dependent bacteriophageDNA infection. J. Mol. Biol. 53:159-162.

[0350] Marugg, J. D., C. F. Gonzalez, B. S. Kunka, A. M. Ledeboer, M. J.Pucci, M. Y. Toonen, S. A. Walker, L. C. M. Zoetmulder, and P. A.Vandenbergh. 1992. Cloning, expression, and nucleotide sequence of genesinvolved in production of pediocin PA-1, a bacteriocin from Pediococcusacidilactici PAC1.0. Appl. Environ. Microbiol. 58:2360-2367.

[0351] McMullen, L. M., and M. E. Stiles. 1993. Microbial ecology offresh pork stored under modified atmosphere at −1, 4.4 and 10° C. Int.J. Food Microbiol. 18:1-14.

[0352] McMullen, L. M., and M. E. Stiles. 1994. Quality of fresh retailpork cuts stored in modified atmosphere under temperature conditionssimulating export to distant markets. Meat Sci. 38:163-177.

[0353] Montel, M.-C., R. Talon, J. Fournaud, and M.-C. Champomier. 1991.A simplified key for identifying homofermentative Lactobacillus andCarnobacterium spp. from meat. J. Appl. Bacteriol. 70: 469-472.

[0354] Muriana, P. M., and T. R. Klaenhammer. 1991. Cloning, phenotypicexpression, and DNA sequence of the gene for Lactacin F, anantimicrobial peptide produced by Lactobacillus spp. Appl. Environ.Microbiol. 173:1779-1788.

[0355] Pearson, W. R., and D. J. Lipman. 1988. Improved tools forbiological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444-2448.

[0356] Pugsley, A. P. 1993. The complete general secretory pathway inGram-negative bacteria. Microbiol. Rev. 57:50-108.

[0357] Quadri, L. E. N., K. L. Roy, J. C. Vederas, and M. E. Stiles.1996. J. Bacteriol. submitted.

[0358] Quadri, L. E. N., M. Sailer, K. L. Roy, J. C. Vederas, and M. E.Stiles. 1994. Chemical and genetic characterization of bacteriocinsproduced by Carnobacterium piscicola LV17B. J. Biol. Chem.269:12204-12211.

[0359] Quadri, L. E. N., M. Sailer, M. Terebiznek, K. L. Roy, J. C.Vederas, and M. E. Stiles. 1995. Characterization of the proteinconferring immunity to the antimicrobial peptide carnobacteriocin B2 andexpression of carnobacteriocins B2 and BM1. J. Bacteriol. 177:1144-1151.

[0360] Randall, L. L., and S. J. S. Hardy. 1986. Correlation ofcompetence for export with lack of tertiary structure of the maturespecies; a study in vivo of maltose binding protein in E. coli. Cell46:921-928.

[0361] Renerre. M. and Montel, M-C. (1986) Inoculation of steaks withLacrobacillus species and effect on colour and microbial counts.Proceedings of the 3'nd meeting of European Meat Research Workers, pp.213-216.

[0362] Reuter, G. 1970. Laktobazillen und eng verwandte Mikroorganismenin Fleisch und Fleischerzeugnissen 2. Mitteilung: Die charakterisierungder isolierten Laktobazillenstämme. Fleischwirtsch. 50: 954-962.

[0363] Rottlander, E., and Trautner, T. A. (1970) Genetic andtransfection studies with Bacillus subtilis phage SP50. J Mol Biol 108:47-60.

[0364] Ruhr, E., and Sahl, H.-G. (1985) Mode of action of the peptideantibiotic nisin and influence on the membrane potential of whole cellsand on cytoplasmic and artificial membrane vesicles. Antimicrob AgentsChemother 27: 841-845.

[0365] Ruiz-Barba, J. .L., D. P. Cathcart, P. J. Warner, and R.Jimenez-Díaz. 1994. Use of Lactobacillus plantarum LPCO10, a bacteriocinproducer, as a starter culture in Spanish-style green olivefermentations. Appl. Environ. Microbiol. 60:2059-2064.

[0366] Sahl, H.-G. (1991) In Nisin and novel lantibiotics. Jung, G. andSahl, H.-G. (eds). Leiden. Escom, pp.347-358.

[0367] Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) MolecularCloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press.

[0368] Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencingwith chain-termination inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467

[0369] SAS Institute (1985) SAS User's Guide: Statistics. SAS Institute,Inc. Cars. NC.

[0370] Schägger, H., and von Jagow, G. (1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Biochem 166: 368-379.

[0371] Schillinger, U. and Lucke, F.-K. (1987) Lactic acid bacteria onvacuum-packed meat and their influence on storage life. Fleischwirtsch.67, 1244-1'48.

[0372] Schillinger, U., and F.-K. Lücke. 1987. Identification oflactobacilli from meat and meat products. Food Microbiol. 4:199-208.

[0373] Schillinger. U. and Lucke, F.-K. (1989) Antibacterial activity ofLacrobacillus sake isolated from meat Appl. Environ. Microbiol. S5,1901-1906.

[0374] Shaw, B. G., and C. D. Harding. 1984. A numerical taxonomic studyof lactic acid bacteria from vacuum packed beef, pork, lamb and bacon.J. Appl. Bacteriol. 56: 25-40.

[0375] Shaw, B. G., and C. D. Harding. 1985. Atypical lactobacilli fromvacuum packaged meats: Comparison by DNA hybridization, cell compositionand biochemical tests with a description of Lactobacillus carnis sp.nov. System. Appl. Microbiol. 6:291-297.

[0376] Shay, B. J. and Egan, A. F. (1981) Hydrogen sulphide productionand spoilage of vacuum-packed beef by a Lactobacillus In: T. A. Roberts,G. Hobbs, J. H. B. Christian and N. Skovgaard (editors), PsychrotrophicMicroorganisms in Spoilage and Pathogenicity. Academic Press. New York.pp. 241-251.

[0377] Simonen, M., and I. Palva. 1993. Protein secretion in Bacillusspecies. Microbiol. Rev. 57:109-137.

[0378] Siragusa and Nettles Cutter ( ) ????

[0379] Smith, G. C., Hall, L. C. and Vanderzant, C. (1980) Inoculationof beef steaks with Lactobacillus species before vacuum packaging. 11.Effect on meat quality characteristics. J. Food Prot. 43, 442-849.

[0380] Southern, E. M. (1975). Detection of specific sequences among DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

[0381] Stiles, M. E. and Hastings, J. W. (1991) Bacteriocin productionby lactic acid bacteria: potential for use in meat preservation. TrendsFood Sci. Technol. 2, 247-251.

[0382] Stoddard, G. W., Petzel, J. P., van Belkum, M. J., Kok, J., andMcKay, L. L. (1992) Molecular analyses of the lactococcin A gene clusterfrom Lactococcus lactis subsp. lactis biovar. diacetylactis WM4. ApplEnviron Microbiol 58: 1952-1961.

[0383] Studier, F. W., and Moffat, B. (1986) Use of bacteriophage T7 RNApolymerase to direct selective high level expression of cloned genes. JMol Biol 189: 113-130.

[0384] Tabor, S., and Richardson, C. C. (1985) A bacteriophage T7 RNApolymerase promoter system for controlled exclusive expression ofspecific genes. Proc Natl Acad Sci USA 82: 1074-1078.

[0385] Terzaghi, B. E., and Sandine, W. E. (1975) Improved medium forlactic streptococci and their bacteriophages. Appl Microbiol 29:807-813.

[0386] Tichaczek, P. S., R. F. Vogel, and W. P. Hammes. 1994. Cloningand sequencing of sakP encoding sakacin P, the bacteriocin produced byLactobacillus sake LTH 673. Microbiology 140:361-367.

[0387] Van Belkum, M. J., B. J. Hayema, R. E Jeeninga, J. Kok, and G.Venema. 1991. Organization and nucleotide sequences of two lactococcalbacteriocin operons. Appl. Environ. Microbiol. 57:492-498.

[0388] Van Belkum, M. J., J. Kok, and G. Venema. 1992. Cloning,sequencing, and expression in Escherichia coli of lcnB, a thirdbacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.Appl. Environ. Microbiol. 58:572-577.

[0389] van Belkum, M. J. 1994. Lactococcins, bacteriocins of Lactococcuslactis, p. 301-318. In L. De Vuyst and E. J. Vandamme (ed.),Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics andApplications. Blackie Academic & Professional, Glasgow.

[0390] van Belkum, M. J., and Stiles, M. E. (1995) Molecularcharacterization of genes involved in the production of the bacteriocinleucocin A from Leuconostoc gelidum. Appl Environ Microbiol 61:3573-3579.

[0391] van Belkum, M. J., Hayema, B. J., Geis, A., Kok, J., and Venema,G. (1989) Cloning of two bacteriocin genes from a lactococcalbacteriocin plasmid. Appl Environ Microbiol 55: 1187-1191.

[0392] van Belkum, M. J., Hayema, B. J., Jeeninga, R. E., Kok, J., andVenema, G. (1991a) Organization and nucleotide sequences of twolactococcal bacteriocin operons. Appl Environ Microbiol 57: 492-498.

[0393] Van Belkum, M. J., J. Kok, and G. Venema. 1992. Cloning,sequencing, and expression in Escherichia coli of lcnB, a thirdbacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.Appl. Environ. Microbiol. 58:572-577.

[0394] van Belkum, M. J., Kok, J., Venema, G., Holo, H., Nes, I. F.,Konings, W. N., and Abee, T. (1991b) The bacteriocin lactococcin Aspecifically increases the permeability of lactococcal cytoplasmicmembranes in a voltage-independent, protein-mediated manner. J Bacteriol173: 7934-7941.

[0395] van de Guchte, M., J. Kok, and G. Venema. 1992. Gene expressionin Lactococcus lactis. FEMS Microbiol. Rev. 88:73-92.

[0396] Van de Guchte, M., J. M. B. M. van der Vossen, J. Kok, and G.Venema. 1989. Construction of a lactococcal expression vector:expression of hen egg white lysozyme in Lactococcus lactis subsp.lactis. Appl. Environ. Microbiol. 55:224-228.

[0397] van der Meer, J. R., Rollema, H. S., Siezen, R. J., Beerthuyzen,M. M., Kuipers, O. P., and de Vos, W. M. (1994) Influence of amino acidsubstitutions in the nisin leader peptide on biosynthesis and secretionof nisin by Lactococcus lactis. J Biol Chem 269: 3555-3562.

[0398] Van der Vossen, J. M. B. M., D.van der Lelie, and G. Venema.1987. Isolation and characterization of Streptococcus cremorisWG2-specific promoters. Appl.Environ. Microbiol. 48:2452-2457.

[0399] van der Vossen, J. M. B. M., J. Kok, and G. Venema (1985).Construction of cloning, promoter-screening, and terminator-screeningshuttle vectors for Bacillus subtilis and Streptococcus lactis. Appl.Environ. Microbiol. 50:540-542. Vandamme (ed.), Bacteriocins of lacticacid bacteria: microbiology, genetics, and applications. Chapman andHall, Ltd. London.

[0400] Venema, K., Dost, M. H. R., Beun, P. A. H., Haandrikman, A. J.,Venema, G., and Kok, J. (1996) The genes for secretion and maturation oflactococcins are located on the chromosome of Lactococcus lactis IL1403.Appl Environ Microbiol 62: 1689-1692.

[0401] Venema, K., Kok, J., Marugg, J. D., Toonen, M. Y., Ledeboer, A.M., Venema, G., and Chikindas, M. L. (1995) Functional analysis of thepediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunitygene and PedD is the processing enzyme. Mol Microbiol 17: 515-522.

[0402] Vieira, J., and J. Messing (1982). The pUC plasmids, anM13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal primers. Gene 19:259-268.

[0403] Vieira, J., and J. Messing. 1987. Production of single-strandedplasmid DNA. Methods Enzymol. 153:3-11.

[0404] Von Heijne, G. 1986. Net N-C charge imbalance may be importantfor signal sequence function in bacteria. J. Mol. Biol. 192:287-290.

[0405] Wandersman, C., and Delepelaire, P. (1990) TolC, an Escherichiacoli outer membrane protein required for hemolysin secretion. Proc NatlAcad Sci USA 87: 4776-4780.

[0406] Wilkinson, B. J., and D. Jones. 1977. A numerical taxonomicsurvey of Listeria and related bacteria. J. Gen. Microbiol. 98: 399-421.

[0407] Worobo, R. W., T. Henkel, M. Sailer, K. L. Roy, J. C. Vederas,and M. E. Stiles. 1994. Characteristics and genetic determinant of ahydrophobic peptide bacteriocin, carnobacteriocin A, produced byCarnobacterium piscicola LV17A. Microbiology 140:517-526.

[0408] Worobo, R. W., M. J. van Belkum, M. Sailer, K. L. Roy, J. C.Vederas, and M. E. Stiles. 1995. A signal peptide secretion-dependentbacteriocin from Carnobacteriurn divergens. J.Bacteriol. 177:3143-3149.

[0409] Zhang, F., Greig, D. I., and Ling, V. (1993) Functionalreplacement of the hemolysin A transport signal by a different primarysequence. Proc Natl Acad Sci USA 90: 4211-4215.

[0410] Zhang, F., Yin, Y., Arrowsmith, C. H., and Ling, V. (1995a)Secretion and circular dichroism analysis of the C-terminal signalpeptides of HlyA and LktA. Biochemistry 34: 4193-4201.

[0411] Zhang, L. H., M. J. Fath, H. K. Mahanty, P. C. Tai, and R.Kolter. 1995b. Genetic analysis of the colicin V secretion pathway.Genetics 141:25-32.

[0412] Zúñiga, M., I. Pardo, and S. Ferrer. 1993. An improved medium fordistinguishing between homofermentative and heterofermentative lacticacid bacteria. Int. J. Food Microbiol. 18:37-42. TABLE 1 Bacterialstrains and plasmids Bacterial Strain or plasmid RelevantCharacteristics^(a) Reference or source Strains C. divergens LV13Leucocin A sensitive indicator strain Shaw^(c) Carnobacteriocinsensitive indicator strain NCFB^(b) dvn + dvi + (containing pCD3.4),CbnB2^(s) (NCFB 2855) AJ Dvn^(s) DbnB2^(r) laboratory isolate C.piscicola LV17C Bac, plasmidless mutant derived from Ahn & Stiles(1990), C.piscicola Lv17B Dvn^(s) DbnB2^(s), plasmidless LV17A cbnA(containing pCp49), Bac⁺Dvn^(s) Ahn & Stiles (1990), LV17B Bac^(F),containing pCP40 Ahn & Stiles (1990), cbnB2 and cbnBMI (containingpCP40) UAL26 Dvn^(s) DbnB2^(s), Bcn⁺, plasmidless, Bac⁺Dvn^(s) Ahn &Stiles (1990), Shaw^(c) Lactococcus lactis subsp. lactis MG1363Dvn^(r),plasmidless Gasson (1983) IL1403 Dvn^(r),DvnB2^(r), plasmidlessChopin et al (1984), Lb. sake 1218 Sulfide producing spoilage organismL. McMullen, U. of Alberta L. gelidum UAL187 bac⁺ wildtype strain with5.0, 7.6 and 9.2 Hastings & Stiles (1991) MDa plasmids UAL187-22 bac⁺strain with 7.6 and 9.2 MDa plasmids Hastings & Stiles (1991) UAL187-13bac strain with 9.2 MDa plasmid Hastings & Stiles (1991) E.Coli DH5α FendAl hsdR17 (r_(k) − m_(k) +)supE44 thi-1 l- BRL Life recA1 gyrA96relA1 (argF- lacZYA)UI69 Techonolgies Inc. f80dlacZ_M15 MH1 MC1061derivative; araD139 lacX74 galU Casadaban & Cohen (1980), galK hsr hsm +strA MV1193 Δ(lac proAB) rpsL thi endA spcB15 hsdR4 Δ(srl-recA)306::Tn10(tetr) F [traD36 proAB⁺ lacl^(q) lacZΔM15] LQ5.21 E. coliMV1193containing pLQ5.21 Quadri et al (1994) LQ7.2 E.coli MV1193 containingpLQ7.2 Quadri et al (1994) Plasmids pCD3.4 dvn+, dvi + (divergicin Aproducer), 3.4 kb Worobo et al (1995) pCD4.4 pCD3.4 containing 1.0-kbEcoRI Cm^(r) gene Worobo et al (1995) of pGS30; Cm^(r)dvn⁺dvi⁺, 4.4 kbpCP40 61-kb plasmid conferring Bac⁺ Imm⁺ Ahn & Stiles (1990) phenotypepGKV210 Em^(r), 4.4 kb van der Vossen et al (1987) pGKV259 Em^(r) Cm^(r)5.0 kb Van der Vossen (1987) pGS30 pUC7 containing 1.0-kb PstI Cm^(r)gene of G. Venema^(d) pC194; Cm^(r), 3.7 kb pJH6.1F pUC118 containing2.9-kb HpaII fragment Hastings et al (1991) from pLG7.6, Amp^(r), 6.1 kbpJKM05 528-bp HindIII-XbaI cbnB2, cbiB2 PCR McCormick et al (1996)product in pUC118, Ampr pJKM07 266-bp EcoR1-IIindIII fragment of pJKM05McCormick et al (1996) in pUC118 pJKM08 262-bp EcoR1 fragment of pJKM05in McCormick et al (1996) pUC118 pJKM14 pMG36e containing divergicin Asignal McCormick et al (1996) peptide fused to carnobacteriocin B2structural gene and also containing carnbacteriocin B2 immunity gene,cbnB2+, cbiB2+, Emr pJKM16 335-bp SacI-EcoR1 fragment from pJKM14McCormick et al (1996) cloned in pUC118 pKMI pUC7 containing 1.3-kb pstIKm^(r) gene of G. Venema^(d) pUB110; Km_(r), 3.7 kb pLG7.6 Lca-Imm⁺, 18kb Hastings & Stiles (1991) pLQ5.21 pUC118 containing a 1.9-kb HindIIIQuadri et al (1995) fragment of pCP40 pLQ7.2 pUC118 containing a 4.0-kbEcoRI-PstI Quadri et al (1995) genomic fragment from C. piscicola LV17CpLQ24 pCaT containing 16-kb insert from pCP40, Quadri et al (1995)cbnB2+, cbiB2+, Cmr, 24.5 kb pMB500 Km^(r), 18.2 kb; specifyinglactococcins van Belkum et al (1989) A and B pMB553 Em^(r), 18.2 kb;specifying lactococcin A van Belkum et al (1989) pMG36e expressionvector, Em^(r), 3.6 kb van Belkum et al (1989) van de Guchte et al(1989) pMJ1 pGKV210 containing 2.9-kb HpaII fragment van Belkum & Stiles(1995) from pJH6.1F, Em^(r), 6.8 kb pMJ3 pGKV210 containing 1-kbHpaI-DraI van Belkum & Stiles (1995) fragment from pJII6.1F, Em^(r), 5.4kb pMJ4 pUC118 containing 12.3-kb HindIII van Belkum & Stiles (1995)fragment from pLG7.6, Amp^(r), 15.5 kb pMJ6 pMG36e containing the 8-kbSacI-HindIII van Belkum & Stiles (1995) fragment from pMJ4. Em^(r), 11.6kb pMJ10 pMG36e containing the 7.9-kb HindIII- van Belkum & Stiles(1995) NruIfragment from pMJ4, Em^(r), 11.4 kb pMJ16 EcoRV-BamHIdeletion derivative of pMJ6, van Belkum & Stiles (1995) Em^(r,), 10.6 kbpMJ17 BstE11-Sta-I deletion derivative of pMJ6. van Belkum & Stiles(1995) Em^(r)m, 10.6 kb pMJ18 EcoRV-HindIII deletion derivative of pMJ6,van Belkum & Stiles (1995) Em^(r), 8.7 kb pMJ20 Frameshift mutation inClaI site of pMJ3, van Belkum & Stiles (1995) Em^(r), 5.4 kb pMJ26Frameshift mutation in NsiI site of pMJ6, van Belkum & Stiles (1995)Em^(r), 11.6 kb pRW19e pMG36e containing 514-bp EcoRV-AccI fragment;dvn+,dvi+, Emr pRW5.6 pGKV259 containing 514-bp EcoRV-AccIfragment;Em^(r)dvn⁺dvi⁺, 5.6 kb pRW6.0 pGKV259 containing divergicinsignal Worobo et al (1995) peptide fused to alkaline phosphatase pUC1183.2-kb cloning vector, Amp^(R),lacZ Veira & Messing (1987) lacZ′ Ampr,3.2 kb Veira & Messing (1987)

[0413] TABLE 2 Spectrum of of Antibiotic Activity of a Variety ofPurified Bacteriocins expressed as the Number of strains inhibited /Number of strains tested Bacteriocin cbn 26 cbn A cbn B Leu A Broch CGenus of Strains tested 1 AU 8 AU 1 AU 8 AU 1 AU 8 AU 1 AU 8 AU 1 AU 8AU Bacillus vegetative cells 2/5 5/5 2/5 2/5 2/5 2/5 2/5 2/5 1/5 4/5spores 5/5 5/5 0/5 0/5 0/8 0/8 0/8 1/8 3/8 3/8 Clostridia vegetativecells 3/8 6/8 0/8 0/8 0/8 0/8 0/8 1/8 3/8 3/8 spores 0/7 4/7 0/7 0/7 0/70/7 0/7 0/7 1/7 1/7 Staphylococcus 1/7 1/7 0/7 1/7 1/7 1/7 0/7 6/7 1/77/7 Streptococcus 2/3 2/3 0/3 0/3 0/3 0/3 0/3 1/3 1/3 2/3 Listeria 42/4242/42  4/42 21/42 10/42 26/42 39/42 40/42  0/42 39/42 G negative  0/29 0/29  0/29  0/29  0/29  0/29  0/29  0/29  0/29  0/29 strainsBrochothrix 14/14 14/14  0/14  0/14  0/14  0/14  0/14  0/14 13/14 13/14Carnobacteria  0/14  0/14  0/14  0/14  0/14  0/14 14/14 14/14  0/14 0/14 Enterococcus 11/14 13/14  2/14  2/14  3/14  3/14  7/14  9/14  8/1412/14 Lactobacillus 15/17 16/17  0/17  1/17  0/17  0/17  1/17  1/17 3/17  8/17 Lactococcus 8/8 8/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 3/8Leuconostoc 9/9 9/9 1/9 1/9 1/9 1/9 5/9 5/9 1/9 8/9 Pediococcus 2/3 3/30/3 0/3 0/3 0/3 0/3 0/3 0/3 2/3

[0414] TABLE 3 Spectrum of of Antibiotic Activity of a Variety ofPurified Bacteriocins expressed as the Number of strains inhibited /Number of strains tested Bacteriocin and Number of units used in theAssay cbn 26 Enterocin Leu A Mesen Y105 Brochocin Nisin Genus of StrainsTested 1 AU 8 AU 1 AU 8 AU 1 AU 8 AU 1 AU 8 AU Bacillus vegetative 2/5 2/5^(a) 3/5 5/5 ^( 3/5) ^(p) 5/5 5/5 5/5 cells spores 0/5 0/5  5/5^(b) 5/5^(b) 5/5 5/5 5/5 5/5 Clostridia vegetative 0/8  1/8^(c) 0/8 1/8^(d) 4/8^(q) 6/8 7/8 8/8 cells spores 0/7 0/7 0/7 0/7 1/7  4/7^(r) 5/7 7/7Staphylococcus  1/7^(e)  1/7^(e)   7/7^(ef)   7/7^(ef) 1/7 7/7 3/7 7/7Streptococcus 0/3 0/3 0/3  3/3^(g) 2/3 2/3 1/3 2/3 Listeria  39/42^(h) 40/42^(i)  36/42^(h) 42/42 42/42 42/42 42/42 42/42 Brochothrix  0/14 0/14  0/14  0/14 14/14 14/14 14/14 14/14 Carnobacteria  12/19^(l) 18/19^(k)  17/19^(l) 19/19 19/19 19/19 19/19 19/19 Enterococcus  7/14^(l)   9/14^(l)   3/14^(l)   9/14^(l) 14/14 14/14 12/14 14/14Lactobacillus    1/17^(m)    1/17^(m)    1/17^(m)    1/17^(m) 15/17 16/17^(s) 16/17 17/17 Lactococcus 0/8 0/8 0/8  1/8^(n) 8/8 8/8 4/8 6/8^(l) Leuconostoc 5/9 5/9 5/9  6/9^(o) 9/9 9/9 9/9 9/9 Pediococcus0/3 0/3 0/3 0/3 2/3 3/3 3/3 3/3

[0415] TABLE 4 Spectrum of of Antibiotic Activity of a Variety ofPurified Bacteriocins expressed as the Number of strains inhibited /Number of strains tested Bacteriocin Tested Mesent Enterocin Y105 PediPA-1 Quest Nisin 900 Genus of Strains tested 1 AU 8 AU 1 AU 8 AU 1 AU 8AU 1 AU 8 AU 1 AU 8 AU Bacillus vegetative cells 3/5 5/5 2/5 2/5 1/5 2/55/5 5/5 0/5 2/5 spores 5/5 5/5 0/5 0/5 0/5 0/5 5/5 5/5 0/5 0/5Clostridia vegetative 0/8 1/8 0/8 0/8 0/8 0/8 7/8 8/8 2/8 3/8 cellsspores 0/7 0/7 0/7 0/7 0/7 0/7 5/7 7/7 0/7 0/7 Staphylococcus 1/7 7/70/7 1/7 0/7 1/7 3/7 7/7 1/7 1/7 Streptococcus 0/3 3/3 0/3 0/3 0/3 0/31/3 2/3 0/3 0/3 Listeria 36/42 42/42 39/42 40/42 38/42 40/42 42/42 42/4239/42 39/42 G negative  0/29  0/29  0/29  0/29  0/29  0/29  0/29  0/29 0/29  0/29 strains Brochothrix  0/14  0/14  0/14  0/14  0/14  0/1414/14 14/14  0/14  0/14 Carnobacteria 17/19 19/19  7/19 10/19  5/19 7/19 19/19 19/19  1/19  7/19 Enterococcus  3/14  9/14  7/14 11/14  1/14 7/14 12/14 14/14  5/14  8/14 Lactobacillus  1/17  2/17  1/17  2/17 1/17  2/17 16/17 17/17  2/17  5/17 Lactococcus 0/8 1/8 0/8 0/8 0/8 0/84/8 6/8 4/8 4/8 Leuconostoc 5/9 6/9 4/9 5/9 1/9 3/9 9/9 9/9 1/9 1/9Pediococcus 0/3 0/3 0/3 2/3 0/3 0/3 3/3 3/3 0/3 0/3

[0416] TABLE 5 Bacteriocin production by Strains of CarnobacteriumIndicator strains^(a) LV17C LV13 MG36e RW19e JKM14 MG36e RW19e JKM14Producer strains C. piscicola LV17C.MG36e 0 0 0 0 0 0 LV17C.RW19e 30 030 0 0 0 LV17C.JKM14 7 6 0 20 20 0 C divergens LV13.MG36e 23 0 23 0 0 0LV13.RW19e 26 0 29 0 0 0 LV13.JKM14 24 6 24 19 19 0

[0417]

1 80 1 184 DNA Divergicin A RBS (24)..(28) 1 atcttggtat cacaaactaatttggaggtt ggtatat atg aaa aaa caa att tta 55 Met Lys Lys Gln Ile Leu 15 aaa ggg ttg gtt ata gtt gtt tgt tta tct ggg gca aca ttt ttc tca 103Lys Gly Leu Val Ile Val Val Cys Leu Ser Gly Ala Thr Phe Phe Ser 10 15 20aca cca caa caa gct tct gct gta aat tat ggt aat ggt gtt tct tgc 151 ThrPro Gln Gln Ala Ser Ala Val Asn Tyr Gly Asn Gly Val Ser Cys 25 30 35 agtaaa aca aaa tgt tca gtt aac tgg gga caa 184 Ser Lys Thr Lys Cys Ser ValAsn Trp Gly Gln 40 45 2 49 PRT Divergicin A misc_feature (114)..(119)label= restriction_site 2 Met Lys Lys Gln Ile Leu Lys Gly Leu Val IleVal Val Cys Leu Ser 1 5 10 15 Gly Ala Thr Phe Phe Ser Thr Pro Gln GlnAla Ser Ala Val Asn Tyr 20 25 30 Gly Asn Gly Val Ser Cys Ser Lys Thr LysCys Ser Val Asn Trp Gly 35 40 45 Gln 3 49 PRT Leuconostoc lactis 3 MetLys Lys Gln Ile Leu Lys Gly Leu Val Ile Val Val Cys Leu Ser 1 5 10 15Gly Ala Thr Phe Phe Ser Thr Pro Gln Gln Ala Ser Ala Val Asn Tyr 20 25 30Gly Asn Gly Val Ser Cys Ser Lys Thr Lys Cys Ser Val Asn Trp Gly 35 40 45Gln 4 4290 DNA Leucocin A gene; 4 aatttttgcc catgctgcat gatattttgactaccaaaaa atatgcgtgt tgcgtacttc 60 aatgttgata atttttttaa agataattcctctgacaaag ctagttatat taatttcttt 120 caagagttaa atatttctca actgccttctttaattttta ctaatggaaa catggactat 180 aaacgattat caatttatac aattaaaacaccaataaatg catggattac tgctattaat 240 gacgaattaa tttcaaaaca ttccaagcaatcatcaacaa attaaaaatg gttaaggtca 300 aaatgtttca aaaaagaata aattatatcgcacaagtaga tgaacgtgat tgtggtgttg 360 ctgcactcgc tatggtttta actcattacaaaacacgcct gtccttagcc aaactacggg 420 acctggccaa aactgacatg gaaggaacgactgctttagg cattgttaaa gctgcgaatg 480 cgctagactt tgaaaccatg ccgatccaggctgatttgag tttattcgat aaaaaggatt 540 taccctatcc ttttatcgcc catgtcattaaagacggtaa atacccgcat tattatgtag 600 tttatgggat caaaggtgat cagctattaatcgctgatcc agataatacc gttggtaaaa 660 ataaaatgac aaaagcgcat tttaatgaggagtggaccgg tgtgtccatt tttattgcgc 720 ccaatccaac ctacaagcca acaaaggataaaaagcgttc cttgacttct tttattccag 780 tgattacgcg tcaaaaatta ttagttatcaatattgtcat tgctgccttg ttggttaccc 840 tagtgagtat tttaggatca tattatttgcaaggtatcat tgatacctat atccccgata 900 acatgaaaaa caccctaggg attgtgtcactagggcttat ttttgcgtat gttatccaac 960 aactgctctc ttatgccaga gattatttattaattgtcat ggggcaacgc ctctcaattg 1020 atattatttt gtcttatatc aaacacatttttgaactgcc aatgtctttt ttcgcgacgc 1080 gtcgtaccgg tgaaattgtg agccgttttacggacgctaa tgccattatt gaagccctgg 1140 caagcacgat gttatctgta tttttagacttaggaatttt ggtcattgtt ggcacagtgc 1200 tagtggttca aaattcaacc ttgtttctgatttctctgat tgccattccg gcttatgccc 1260 tagtggtctg gctctttatg cgtcctttttcaaagatgaa taatgaccaa atgcaagcag 1320 gttcgatgtt aagttcttcc attattgaagatattaatgg cgttgagacg attaaagcgc 1380 tgaatagtga agaaaccgcc tatcataaaattgatcatga atttgtcact tatttagaaa 1440 aatcatttgt ttacgctaaa acagaagccactcaaaatgc gattaaaagc ctcttacagc 1500 tctctttaaa tgtcgtgatc ttatgggttggcgcacaact ggtcatgacc aataaaatta 1560 gtgttggtca actgatcact tacaatgctttattaggatt ttttacagat cccttgcaaa 1620 atattattaa tttacaaact aagctccaacaggcctcagt cgctaataat cgtttgaacg 1680 aagtttattt ggttgattca gaatttaaagctagtcatca aatgacagaa agcattatgc 1740 ccaatagctc attagtagcc gatcatatcacctataaata cggttttggt gcgccagcaa 1800 ttgatgatgt ttcactaacg attacagccggtgaaaaaat cgctttggtt gggattagtg 1860 gatcaggtaa atcaacttta gttaaattgctggttaattt ctttcaacca gagtcaggga 1920 caatttcact aggacaaaca ccactcgccaatcttgataa acatgagcta agagcacaca 1980 ttaattattt accacaagaa ccctttatattttccggttc aattatggac aacctgttat 2040 tgggggctaa gccagggaca acccaagaagatattatcag ggcggtagaa attgctgaaa 2100 ttaaagatga tattgaaaaa atgtcgcaaggatttggcac tgaactcgca gaaagtggca 2160 atatttcggg tggtcaaaaa caacgcattgctttagctag agccatttta gtcgattctc 2220 cggtgctgat tttagatgag tcaaccagtaatcttgatgt tttaacagaa aaaaagatta 2280 ttgataatct catgcagtta accgaaaaaaccattatctt tgtagcgcac cgcttaacca 2340 tttcacagcg agtagatcgt attctaaccatgcaaaacgg caaaattatc gaagatggca 2400 cgcataatac tctgcttaat gccggtggtttctacgcgtc attgtttaat cattaaggag 2460 acctgatgtt tgatccaaaa tacttagaaagtggcgaatt ttatcaacgt cgttaccgca 2520 attttccaac tctgattatt gtgcctatttttttgttagt cgtgtttatc attctattta 2580 gcctatttgc taagcgtgaa attgttgtcaaagcaagtgg cgaaattatt ccagccaaag 2640 tgctatcaga tatccaatca accagtaacaatgccatcga tagtaaccaa ttaactgaaa 2700 ataaagtggt taaaaaaggc gataccttagtgacctttac cagtggtaat gaaaaaatat 2760 cgtctcaatt actgacgcaa caacttaataatcttaacga ccgtctaaaa agtcttgata 2820 cctataagca gagtattgtt aacggacgtagcgaatttgg tggcacagat caatttggtt 2880 atgatagtct attcaacggc tatatggcgcaagttgatac gttgacgagt gaatttaatc 2940 aacaaagtag tgataaacaa acagctgatcaacaagctaa tcatcaaatt gacgttttaa 3000 aacaaggtca atctaaaaac aatcaacaattagctaatta tcaagctatt ctaaccagta 3060 ttaatagcaa cactaaaccg actaataatccctatcaagc catttatgat aattattcag 3120 cccagttaaa atcagcacaa acaactgatgataaagatca agtcaagcaa actgccttaa 3180 gtaatgtaca acaacaaatt gatcaattacaaacaacgag tagttcgtat gatagtcaaa 3240 ttgctggtat tacaaagagt ggtcctttatctcaaagcag taccttagat aaaatcgctg 3300 acttgaagca acaacaacta gcgagtgctcaaaaagaaat caatgatcag caacaatcct 3360 tagatgagtt aaaagccaag caatcctctgctaatgagga ttatcaagat acggttatta 3420 aagcaccaga agatggcatt ttacatttagccactgacaa aactaaaatc aagtatttcc 3480 ctaaaggcac aaccattgcg caaatttatcctaaactgac gcaaaaaaca gctttgaatg 3540 ttgagtacta tgtgcctgcc agtaatattatcggcttaaa gcaaagacaa gccatccgtt 3600 ttgtagcaaa tcaaaatgtc acgaaaccgctcaccttaaa cggaacaatc aaaagcatta 3660 gttctgcacc aatagccagt aaagagggatccttttataa attagtcgcg acgattcagg 3720 ctagcaaaat agaccgtgaa cagattaaatatggtcttaa tggtcgaatc acaaccataa 3780 aagggactaa aacatggttt aattattataaagacattgt tttaggtgag aataattagc 3840 taggaagata aacacaattt ttaaacgtgtttatcttttt tagtctcaat gaaattgtcg 3900 ccgaaggttt ttctagccaa gtggcaggacacagaaaaat gatagttgct actgaaggga 3960 agttcaactg ccaccaaaaa tagtaaccgcgcgacagcca accgccacca caacagttat 4020 gctcgcccgt ggttattatt atcattaacactcttacgtc tttctatgat acttttgagc 4080 cacattctta taatgctgca atcgaccttttagaaaattg atctcatcag aaatttcttt 4140 taagtggtta tcatcagcat gtttactagcaatatttaat tctttaatcc tacgtttaat 4200 caacttagta gttttagtat ctttcatgtattgattatct caaaaaaaca cccaacaagg 4260 gcaatcagtt tgatttgagc agaggaagcc4290 5 717 PRT lcaC; 5 Met Phe Gln Lys Arg Ile Asn Tyr Ile Ala Gln ValAsp Glu Arg Asp 1 5 10 15 Cys Gly Val Ala Ala Leu Ala Met Val Leu ThrHis Tyr Lys Thr Arg 20 25 30 Leu Ser Leu Ala Lys Leu Arg Asp Leu Ala LysThr Asp Met Glu Gly 35 40 45 Thr Thr Ala Leu Gly Ile Val Lys Ala Ala AsnAla Leu Asp Phe Glu 50 55 60 Thr Met Pro Ile Gln Ala Asp Leu Ser Leu PheAsp Lys Lys Asp Leu 65 70 75 80 Pro Tyr Pro Phe Ile Ala His Val Ile LysAsp Gly Lys Tyr Pro His 85 90 95 Tyr Tyr Val Val Tyr Gly Ile Lys Gly AspGln Leu Leu Ile Ala Asp 100 105 110 Pro Asp Asn Thr Val Gly Lys Asn LysMet Thr Lys Ala His Phe Asn 115 120 125 Glu Glu Trp Thr Gly Val Ser IlePhe Ile Ala Pro Asn Pro Thr Tyr 130 135 140 Lys Pro Thr Lys Asp Lys LysArg Ser Leu Thr Ser Phe Ile Pro Val 145 150 155 160 Ile Thr Arg Gln LysLeu Leu Val Ile Asn Ile Val Ile Ala Ala Leu 165 170 175 Leu Val Thr LeuVal Ser Ile Leu Gly Ser Tyr Tyr Leu Gln Gly Ile 180 185 190 Ile Asp ThrTyr Ile Pro Asp Asn Met Lys Asn Thr Leu Gly Ile Val 195 200 205 Ser LeuGly Leu Ile Phe Ala Tyr Val Ile Gln Gln Leu Leu Ser Tyr 210 215 220 AlaArg Asp Tyr Leu Leu Ile Val Met Gly Gln Arg Leu Ser Ile Asp 225 230 235240 Ile Ile Leu Ser Tyr Ile Lys His Ile Phe Glu Leu Pro Met Ser Phe 245250 255 Phe Ala Thr Arg Arg Thr Gly Glu Ile Val Ser Arg Phe Thr Asp Ala260 265 270 Asn Ala Ile Ile Glu Ala Leu Ala Ser Thr Met Leu Ser Val PheLeu 275 280 285 Asp Leu Gly Ile Leu Val Ile Val Gly Thr Val Leu Val ValGln Asn 290 295 300 Ser Thr Leu Phe Leu Ile Ser Leu Ile Ala Ile Pro AlaTyr Ala Leu 305 310 315 320 Val Val Trp Leu Phe Met Arg Pro Phe Ser LysMet Asn Asn Asp Gln 325 330 335 Met Gln Ala Gly Ser Met Leu Ser Ser SerIle Ile Glu Asp Ile Asn 340 345 350 Gly Val Glu Thr Ile Lys Ala Leu AsnSer Glu Glu Thr Ala Tyr His 355 360 365 Lys Ile Asp His Glu Phe Val ThrTyr Leu Glu Lys Ser Phe Val Tyr 370 375 380 Ala Lys Thr Glu Ala Thr GlnAsn Ala Ile Lys Ser Leu Leu Gln Leu 385 390 395 400 Ser Leu Asn Val ValIle Leu Trp Val Gly Ala Gln Leu Val Met Thr 405 410 415 Asn Lys Ile SerVal Gly Gln Leu Ile Thr Tyr Asn Ala Leu Leu Gly 420 425 430 Phe Phe ThrAsp Pro Leu Gln Asn Ile Ile Asn Leu Gln Thr Lys Leu 435 440 445 Gln GlnAla Ser Val Ala Asn Asn Arg Leu Asn Glu Val Tyr Leu Val 450 455 460 AspSer Glu Phe Lys Ala Ser His Gln Met Thr Glu Ser Ile Met Pro 465 470 475480 Asn Ser Ser Leu Val Ala Asp His Ile Thr Tyr Lys Tyr Gly Phe Gly 485490 495 Ala Pro Ala Ile Asp Asp Val Ser Leu Thr Ile Thr Ala Gly Glu Lys500 505 510 Ile Ala Leu Val Gly Ile Ser Gly Ser Gly Lys Ser Thr Leu ValLys 515 520 525 Leu Leu Val Asn Phe Phe Gln Pro Glu Ser Gly Thr Ile SerLeu Gly 530 535 540 Gln Thr Pro Leu Ala Asn Leu Asp Lys His Glu Leu ArgAla His Ile 545 550 555 560 Asn Tyr Leu Pro Gln Glu Pro Phe Ile Phe SerGly Ser Ile Met Asp 565 570 575 Asn Leu Leu Leu Gly Ala Lys Pro Gly ThrThr Gln Glu Asp Ile Ile 580 585 590 Arg Ala Val Glu Ile Ala Glu Ile LysAsp Asp Ile Glu Lys Met Ser 595 600 605 Gln Gly Phe Gly Thr Glu Leu AlaGlu Ser Gly Asn Ile Ser Gly Gly 610 615 620 Gln Lys Gln Arg Ile Ala LeuAla Arg Ala Ile Leu Val Asp Ser Pro 625 630 635 640 Val Leu Ile Leu AspGlu Ser Thr Ser Asn Leu Asp Val Leu Thr Glu 645 650 655 Lys Lys Ile IleAsp Asn Leu Met Gln Leu Thr Glu Lys Thr Ile Ile 660 665 670 Phe Val AlaHis Arg Leu Thr Ile Ser Gln Arg Val Asp Arg Ile Leu 675 680 685 Thr MetGln Asn Gly Lys Ile Ile Glu Asp Gly Thr His Asn Thr Leu 690 695 700 LeuAsn Ala Gly Gly Phe Tyr Ala Ser Leu Phe Asn His 705 710 715 6 457 PRTlcaD; 6 Met Phe Asp Pro Lys Tyr Leu Glu Ser Gly Glu Phe Tyr Gln Arg Arg1 5 10 15 Tyr Arg Asn Phe Pro Thr Leu Ile Ile Val Pro Ile Phe Leu LeuVal 20 25 30 Val Phe Ile Ile Leu Phe Ser Leu Phe Ala Lys Arg Glu Ile ValVal 35 40 45 Lys Ala Ser Gly Glu Ile Ile Pro Ala Lys Val Leu Ser Asp IleGln 50 55 60 Ser Thr Ser Asn Asn Ala Ile Asp Ser Asn Gln Leu Thr Glu AsnLys 65 70 75 80 Val Val Lys Lys Gly Asp Thr Leu Val Thr Phe Thr Ser GlyAsn Glu 85 90 95 Lys Ile Ser Ser Gln Leu Leu Thr Gln Gln Leu Asn Asn LeuAsn Asp 100 105 110 Arg Leu Lys Ser Leu Asp Thr Tyr Lys Gln Ser Ile ValAsn Gly Arg 115 120 125 Ser Glu Phe Gly Gly Thr Asp Gln Phe Gly Tyr AspSer Leu Phe Asn 130 135 140 Gly Tyr Met Ala Gln Val Asp Thr Leu Thr SerGlu Phe Asn Gln Gln 145 150 155 160 Ser Ser Asp Lys Gln Thr Ala Asp GlnGln Ala Asn His Gln Ile Asp 165 170 175 Val Leu Lys Gln Gly Gln Ser LysAsn Asn Gln Gln Leu Ala Asn Tyr 180 185 190 Gln Ala Ile Leu Thr Ser IleAsn Ser Asn Thr Lys Pro Thr Asn Asn 195 200 205 Pro Tyr Gln Ala Ile TyrAsp Asn Tyr Ser Ala Gln Leu Lys Ser Ala 210 215 220 Gln Thr Thr Asp AspLys Asp Gln Val Lys Gln Thr Ala Leu Ser Asn 225 230 235 240 Val Gln GlnGln Ile Asp Gln Leu Gln Thr Thr Ser Ser Ser Tyr Asp 245 250 255 Ser GlnIle Ala Gly Ile Thr Lys Ser Gly Pro Leu Ser Gln Ser Ser 260 265 270 ThrLeu Asp Lys Ile Ala Asp Leu Lys Gln Gln Gln Leu Ala Ser Ala 275 280 285Gln Lys Glu Ile Asn Asp Gln Gln Gln Ser Leu Asp Glu Leu Lys Ala 290 295300 Lys Gln Ser Ser Ala Asn Glu Asp Tyr Gln Asp Thr Val Ile Lys Ala 305310 315 320 Pro Glu Asp Gly Ile Leu His Leu Ala Thr Asp Lys Thr Lys IleLys 325 330 335 Tyr Phe Pro Lys Gly Thr Thr Ile Ala Gln Ile Tyr Pro LysLeu Thr 340 345 350 Gln Lys Thr Ala Leu Asn Val Glu Tyr Tyr Val Pro AlaSer Asn Ile 355 360 365 Ile Gly Leu Lys Gln Arg Gln Ala Ile Arg Phe ValAla Asn Gln Asn 370 375 380 Val Thr Lys Pro Leu Thr Leu Asn Gly Thr IleLys Ser Ile Ser Ser 385 390 395 400 Ala Pro Ile Ala Ser Lys Glu Gly SerPhe Tyr Lys Leu Val Ala Thr 405 410 415 Ile Gln Ala Ser Lys Ile Asp ArgGlu Gln Ile Lys Tyr Gly Leu Asn 420 425 430 Gly Arg Ile Thr Thr Ile LysGly Thr Lys Thr Trp Phe Asn Tyr Tyr 435 440 445 Lys Asp Ile Val Leu GlyGlu Asn Asn 450 455 7 35 PRT Divergicin N-terminal; Cleavage_site(29)..(30) 7 Met Lys Lys Gln Ile Leu Lys Gly Leu Val Ile Val Val Cys LeuSer 1 5 10 15 Gly Ala Thr Phe Phe Ser Thr Pro Gln Gln Ala Ser Ala AlaAla Pro 20 25 30 Lys Ile Thr 35 8 29 PRT Divergicin N-terminal; 8 MetLys Lys Gln Ile Leu Lys Gly Leu Val Ile Val Val Cys Leu Ser 1 5 10 15Gly Ala Thr Phe Phe Ser Thr Pro Gln Gln Ala Ser Ala 20 25 9 30 PRTLeucocin N-terminal; cleavage_site (24)..(25) 9 Met Met Asn Met Lys ProThr Glu Ser Tyr Glu Gln Leu Asp Asn Ser 1 5 10 15 Ala Leu Glu Gln ValVal Gly Gly Lys Tyr Tyr Gly Asn Gly 20 25 30 10 24 PRT LeucocinN-terminal; 10 Met Met Asn Met Lys Pro Thr Glu Ser Tyr Glu Gln Leu AspAsn Ser 1 5 10 15 Ala Leu Glu Gln Val Val Gly Gly 20 11 27 PRTLactococcin A N-terminal; cleavage_site (21)..(22) 11 Met Lys Asn GlnLeu Asn Phe Asn Ile Val Ser Asp Glu Glu Leu Ser 1 5 10 15 Glu Ala AsnGly Gly Lys Leu Thr Phe Ile Gln 20 25 12 21 PRT Lactococcin A; 12 MetLys Asn Gln Leu Asn Phe Asn Ile Val Ser Asp Glu Glu Leu Ser 1 5 10 15Glu Ala Asn Gly Gly 20 13 21 PRT colicin V N-terminal; cleavage_site(15)..(16) 13 Met Arg Thr Leu Thr Leu Asn Glu Leu Asp Ser Val Ser GlyGly Ala 1 5 10 15 Ser Gly Arg Asp Ile 20 14 15 PRT colicin V; 14 Met ArgThr Leu Thr Leu Asn Glu Leu Asp Ser Val Ser Gly Gly 1 5 10 15 15 3475DNA plasmid; 15 gatatcttgg tattacaaac taattggagg ttggtatata tgaaaaaacaaattttaaaa 60 gggttggtta tagttgtttg tttatctggg gcaacatttt tctcaacaccacaacaagct 120 tctgctgctg caccgaaaat tactcaaaaa caaaaaaatt gtgttaatggacaattaggt 180 ggaatgcttg ctggagcttt gggtggacct ggcggagttg tgttaggtggtataggtggt 240 gcaatagcag gaggttgttt taattaaatg aaaataaaat ggtactgggaatctctgatt 300 gaaaccttaa tatttataat tgttcttctt gtattttttt atagaagttctggtttttct 360 ttaaaaaatt tagttttagg aagtttattt tatttgatag caattggtctttttaattat 420 aaaaagataa acaaataggc actattttta aatttacaac ttttgcattttaagtatatt 480 gttgttatta ttaaggtgcg agatgagata aggtctacat ggacagcacaaaacccaccc 540 ctaatgcgaa taggggtggg tttttttcgt tcgttgcgaa tacgaacgtgtgggttagag 600 acaacttgcg agattatcgt ctaatcatct aaccaatgat ccactagtattaatactagt 660 cccacaaaaa gtggagcaat aaccaatgag ataaggtttt ccataaacagcacccccttt 720 caggggcaag ttgccactta ctaatatagc acagctcctt tattgttcttagtctaaatc 780 tgataaatct tttcttgttc aaaaatatag accacttaaa agcttataacggtactagat 840 ttttcagata ccccaattac ctacttaaaa cgtctctctt tttcgttttaagatgtttaa 900 aattattttc tatgaattat acacaaatgt gcttaaatcg tcttaaatcgtcttaaaatg 960 tggtctgtgt tgagaataca acgactttgt ttggtcgtac ctctaaatctgtttgctgtg 1020 aacgagggta gcgaagtgaa ctttttgttg ctaacgctct tggttttgtcttttgatttt 1080 ataaaatgtg gatgtaatcc actccttact aggggtttaa tctttataaaataaaggagc 1140 ttgcgaatgc aaggtgccct tttttctttg tctgactact agggacaaattatctgagta 1200 tgaacaagat tttgtctgtt cttgcgcgta tttattaata tatattttaagagatatttt 1260 aagagatatt ttaaaacctt tttaggggtg agctcagcct tagagagagtaagcattgaa 1320 gcatagtact agggacaaat tatctgacta ctagggataa attatctgactactagggac 1380 aaattatctg actactaggg ataaattatc tgactactag ggacgcactttactttgtgt 1440 atcgtatcgt ttataatctt tatatgtgag gggaggtcga aaggattggaaaagaaaacg 1500 aatttaaaaa ttgcatatca aaatgaattg aatctggttc cacttaaaaatttcaatgct 1560 aaacaaatgg atttattctt tgctttgtgt gcccgaatga aaaataaagggcttagaaag 1620 gtatctttta cgtttgaaga actaagagaa ctaagtgatt acaagataactgctatcgaa 1680 ccgttcacga atgatttaga acaattatac aaaaaaatgt taaacctaacatacagaacg 1740 gagacagaaa caaaaatcag ttatttcgtt ttatttactg ggtttgtgattgataaatca 1800 gagcaaattg ttgaagttag tgtaaaccca gacttagaac atatcattaacggtatctct 1860 agtgagttca ataaatttga gttactagca ttcacaagta tccagtcgaaatatacgaaa 1920 acactcttta gattgcttat gcagtttcaa tcaactgggt tttatgtggttaaaattgaa 1980 gatttcagag cacttttaga cattccaaaa tcttatcaaa tgactgacataacccaacgg 2040 atattgaaac ctagtttaat tgagttaagt cagtacttta atgatttaaaagttaataaa 2100 attaaagctc gaaagggtaa taaaatagac cgtttagaat tcactttctccggtctaaag 2160 actgatttac ctaaagttcc attgcacgac tggacgaaat aaaaaaaggacctccccctc 2220 acatttaagc aagtaggaac gtccctcgca atccacgaag actgctgattcattttagca 2280 tatattgtgc gggacttcta aataaattat atttggaggt catttttatgtcgaataagt 2340 acttgaaaaa aagaaagcgt caagctaagc aggtagctga tttgtacgatttaattattg 2400 gggttgaaca tgctggcagc tcgttaattg cgttgtatga gggaattaaaccctctcaat 2460 atcgaatttt tattcttttg tcttattcta gttttgaaaa taaattaaacttatacaaca 2520 aagcgatttt aagaactgaa gtttattctt tagaaaaaaa attaaacgaaaaaataaatg 2580 ctcaaatcag aattgcgcaa aaaaataaaa aagaaattgc ggtaattgatttcacaaaac 2640 aaaaagaaaa actcaaaaga gaattactta gttttgaaaa tgataaagaaatgaaactta 2700 tggattcgca attaaaacaa tttcatgaaa ataaaacgtt agctgatattaatgatcagt 2760 tttttatgac ggtacaaaat agtttaattt tgctgcataa aaaagcacctttaacattaa 2820 aattaatttg tttgaaaaat tatattcgcc tttgcaaaaa ttattttctaaagaatatat 2880 tttaatgttt tttgaaaaaa atagtaacat gggaacatgt tgctctgctcgcaaaaggaa 2940 aaatatttaa actaataaaa aaccgtcgga gaccagccaa ccaataggttggctttaagt 3000 ttaagcctac gttgacaact gtcaatgtat aagtgcgccc tttgggtgttttattttttg 3060 tttaactatt attttctgca taggtttttt atttttatta atttgattttcaagaaaggg 3120 atgaacctaa aatgatttat aaacaaaaaa agaaagaaga tgtttttggatttcctaaag 3180 ttttaacaat tgctgatttg agtacgagat ggaaaatgtc acgtcaagctatccataaaa 3240 aaattcaaga agatttatta tttcctatgc ctgttcaaat tgtctcaaatggaaaaatta 3300 aattgttttt atttgttgat attgaaaaat acgaaaaaaa tcgtccgtggttattagaca 3360 ttaattatcg aaatgaacga caactttgga tttacaaaaa tggtttttttaaatagcaag 3420 ttagtcaatt accttatacc ttgttggata tctttggata aaaaaatagttgtat 3475 16 227 DNA Divergicin structural gene; CDS (1)..(225) 16 atgaaa aaa caa att tta aaa ggg ttg gtt ata gtt gtt tgt tta tct 48 Met LysLys Gln Ile Leu Lys Gly Leu Val Ile Val Val Cys Leu Ser 1 5 10 15 ggggca aca ttt ttc tca aca cca caa caa gct tct gct gct gca ccg 96 Gly AlaThr Phe Phe Ser Thr Pro Gln Gln Ala Ser Ala Ala Ala Pro 20 25 30 aaa attact caa aaa caa aaa aat tgt gtt aat gga caa tta ggt gga 144 Lys Ile ThrGln Lys Gln Lys Asn Cys Val Asn Gly Gln Leu Gly Gly 35 40 45 atg ctt gctgga gct ttg ggt gga cct ggc gga gtt gtg tta ggt ggt 192 Met Leu Ala GlyAla Leu Gly Gly Pro Gly Gly Val Val Leu Gly Gly 50 55 60 ata ggt ggt gcaata gca gga ggt tgt ttt aat ta 227 Ile Gly Gly Ala Ile Ala Gly Gly CysPhe Asn 65 70 75 17 75 PRT Divergicin structural gene; 17 Met Lys LysGln Ile Leu Lys Gly Leu Val Ile Val Val Cys Leu Ser 1 5 10 15 Gly AlaThr Phe Phe Ser Thr Pro Gln Gln Ala Ser Ala Ala Ala Pro 20 25 30 Lys IleThr Gln Lys Gln Lys Asn Cys Val Asn Gly Gln Leu Gly Gly 35 40 45 Met LeuAla Gly Ala Leu Gly Gly Pro Gly Gly Val Val Leu Gly Gly 50 55 60 Ile GlyGly Ala Ile Ala Gly Gly Cys Phe Asn 65 70 75 18 75 PRT Divergicinstructural gene 18 Met Lys Lys Gln Ile Leu Lys Gly Leu Val Ile Val ValCys Leu Ser 1 5 10 15 Gly Ala Thr Phe Phe Ser Thr Pro Gln Gln Ala SerAla Ala Ala Pro 20 25 30 Lys Ile Thr Gln Lys Gln Lys Asn Cys Val Asn GlyGln Leu Gly Gly 35 40 45 Met Leu Ala Gly Ala Leu Gly Gly Pro Gly Gly ValVal Leu Gly Gly 50 55 60 Ile Gly Gly Ala Ile Ala Gly Gly Cys Phe Asn 6570 75 19 170 DNA divergicin immunity gene; CDS (1)..(168) 19 atg aaa ataaaa tgg tac tgg gaa tct ctg att gaa acc tta ata ttt 48 Met Lys Ile LysTrp Tyr Trp Glu Ser Leu Ile Glu Thr Leu Ile Phe 1 5 10 15 ata att gttctt ctt gta ttt ttt tat aga agt tct ggt ttt tct tta 96 Ile Ile Val LeuLeu Val Phe Phe Tyr Arg Ser Ser Gly Phe Ser Leu 20 25 30 aaa aat tta gtttta gga agt tta ttt tat ttg ata gca att ggt ctt 144 Lys Asn Leu Val LeuGly Ser Leu Phe Tyr Leu Ile Ala Ile Gly Leu 35 40 45 ttt aat tat aaa aagata aac aaa ta 170 Phe Asn Tyr Lys Lys Ile Asn Lys 50 55 20 56 PRTdivergicin immunity gene; 20 Met Lys Ile Lys Trp Tyr Trp Glu Ser Leu IleGlu Thr Leu Ile Phe 1 5 10 15 Ile Ile Val Leu Leu Val Phe Phe Tyr ArgSer Ser Gly Phe Ser Leu 20 25 30 Lys Asn Leu Val Leu Gly Ser Leu Phe TyrLeu Ile Ala Ile Gly Leu 35 40 45 Phe Asn Tyr Lys Lys Ile Asn Lys 50 5521 56 PRT Divergicin immunity gene 21 Met Lys Ile Lys Trp Tyr Trp GluSer Leu Ile Glu Thr Leu Ile Phe 1 5 10 15 Ile Ile Val Leu Leu Val PhePhe Tyr Arg Ser Ser Gly Phe Ser Leu 20 25 30 Lys Asn Leu Val Leu Gly SerLeu Phe Tyr Leu Ile Ala Ile Gly Leu 35 40 45 Phe Asn Tyr Lys Lys Ile AsnLys 50 55 22 124 DNA Divergicin signal peptide; sig_peptide (1)..(123)22 atc ttg gta tca caa act aat ttg gag gtt ggt ata tat gaa aaa aca 48Ile Leu Val Ser Gln Thr Asn Leu Glu Val Gly Ile Tyr Glu Lys Thr 1 5 1015 aat ttt aaa agg gtt ggt tat agt tgt ttg ttt atc tgg ggc aac att 96Asn Phe Lys Arg Val Gly Tyr Ser Cys Leu Phe Ile Trp Gly Asn Ile 20 25 30ttt ctc aac acc aca aca agc ttc tgc t 124 Phe Leu Asn Thr Thr Thr SerPhe Cys 35 40 23 41 PRT Divergicin signal peptide; 23 Ile Leu Val SerGln Thr Asn Leu Glu Val Gly Ile Tyr Glu Lys Thr 1 5 10 15 Asn Phe LysArg Val Gly Tyr Ser Cys Leu Phe Ile Trp Gly Asn Ile 20 25 30 Phe Leu AsnThr Thr Thr Ser Phe Cys 35 40 24 41 PRT divergicin signal peptide 24 IleLeu Val Ser Gln Thr Asn Leu Glu Val Gly Ile Tyr Glu Lys Thr 1 5 10 15Asn Phe Lys Arg Val Gly Tyr Ser Cys Leu Phe Ile Trp Gly Asn Ile 20 25 30Phe Leu Asn Thr Thr Thr Ser Phe Cys 35 40 25 675 DNA Brochocin-C; 25gctattttga gaaatattaa ccaatagtaa aaattatcat gctatctttt gtatgtaata 60aaaattattt aaaggagggt gtttcatcat gcacaaggta aaaaaattaa acaatcaaga 120gttacaacag atcgtgggag gttacagttc aaaagattgt ctaaaagata ttggtaaagg 180aattggtgct ggtacagtag ctggggcagc cggcggtggc ctagctgcag gattaggtgc 240tatcccagga gcattcgttg gagcacattt tggagtaatc ggcggatctg ccgcatgcat 300tggtggatta ttaggtaact aggaggttat atttatgaaa aaagaactat tgaataaaaa 360tgaaatgagt agaattatcg gcggcaaaat aaattgggga aatgttggcg gttcttgtgt 420tggaggtgca gtaattggag gcgccctcgg tggactaggt ggagctggcg gaggttgcat 480tacaggagct atcggaagta tttgggatca atggtaaaaa ctatactatt ttcggttgta 540atttcattcg ttgcattatg taacttttta ataaaaaaag atgtgtcttc aaaaaaaaaa 600ttatttttaa caggttctat tgctgtcttt ctaattatct atgattttct atggattata 660ttctctaact agtac 675 26 233 DNA Brochocin-C peptide A; CDS (1)..(231) 26atg cac aag gta aaa aaa tta aac aat caa gag tta caa cag atc gtg 48 MetHis Lys Val Lys Lys Leu Asn Asn Gln Glu Leu Gln Gln Ile Val 1 5 10 15gga ggt tac agt tca aaa gat tgt cta aaa gat att ggt aaa gga att 96 GlyGly Tyr Ser Ser Lys Asp Cys Leu Lys Asp Ile Gly Lys Gly Ile 20 25 30 ggtgct ggt aca gta gct ggg gca gcc ggc ggt ggc cta gct gca gga 144 Gly AlaGly Thr Val Ala Gly Ala Ala Gly Gly Gly Leu Ala Ala Gly 35 40 45 tta ggtgct atc cca gga gca ttc gtt gga gca cat ttt gga gta atc 192 Leu Gly AlaIle Pro Gly Ala Phe Val Gly Ala His Phe Gly Val Ile 50 55 60 ggc gga tctgcc gca tgc att ggt gga tta tta ggt aac ta 233 Gly Gly Ser Ala Ala CysIle Gly Gly Leu Leu Gly Asn 65 70 75 27 77 PRT Brochocin-C peptide A; 27Met His Lys Val Lys Lys Leu Asn Asn Gln Glu Leu Gln Gln Ile Val 1 5 1015 Gly Gly Tyr Ser Ser Lys Asp Cys Leu Lys Asp Ile Gly Lys Gly Ile 20 2530 Gly Ala Gly Thr Val Ala Gly Ala Ala Gly Gly Gly Leu Ala Ala Gly 35 4045 Leu Gly Ala Ile Pro Gly Ala Phe Val Gly Ala His Phe Gly Val Ile 50 5560 Gly Gly Ser Ala Ala Cys Ile Gly Gly Leu Leu Gly Asn 65 70 75 28 77PRT brochocin C, peptide A 28 Met His Lys Val Lys Lys Leu Asn Asn GlnGlu Leu Gln Gln Ile Val 1 5 10 15 Gly Gly Tyr Ser Ser Lys Asp Cys LeuLys Asp Ile Gly Lys Gly Ile 20 25 30 Gly Ala Gly Thr Val Ala Gly Ala AlaGly Gly Gly Leu Ala Ala Gly 35 40 45 Leu Gly Ala Ile Pro Gly Ala Phe ValGly Ala His Phe Gly Val Ile 50 55 60 Gly Gly Ser Ala Ala Cys Ile Gly GlyLeu Leu Gly Asn 65 70 75 29 182 DNA Brochocin-C peptide B; CDS(1)..(177) 29 atg aaa aaa gaa cta ttg aat aaa aat gaa atg agt aga attatc ggc 48 Met Lys Lys Glu Leu Leu Asn Lys Asn Glu Met Ser Arg Ile IleGly 1 5 10 15 ggc aaa ata aat tgg gga aat gtt ggc ggt tct tgt gtt ggaggt gca 96 Gly Lys Ile Asn Trp Gly Asn Val Gly Gly Ser Cys Val Gly GlyAla 20 25 30 gta att gga ggc gcc ctc ggt gga cta ggt gga gct ggc gga ggttgc 144 Val Ile Gly Gly Ala Leu Gly Gly Leu Gly Gly Ala Gly Gly Gly Cys35 40 45 att aca gga gct atc gga agt att tgg gat caa tggta 182 Ile ThrGly Ala Ile Gly Ser Ile Trp Asp Gln 50 55 30 59 PRT Brochocin-C peptideB; 30 Met Lys Lys Glu Leu Leu Asn Lys Asn Glu Met Ser Arg Ile Ile Gly 15 10 15 Gly Lys Ile Asn Trp Gly Asn Val Gly Gly Ser Cys Val Gly Gly Ala20 25 30 Val Ile Gly Gly Ala Leu Gly Gly Leu Gly Gly Ala Gly Gly Gly Cys35 40 45 Ile Thr Gly Ala Ile Gly Ser Ile Trp Asp Gln 50 55 31 60 PRTBrochocin-C, peptide B 31 Met Lys Lys Glu Leu Leu Asn Lys Asn Glu MetSer Arg Ile Ile Gly 1 5 10 15 Gly Lys Ile Asn Trp Gly Asn Val Gly GlySer Cys Val Gly Gly Ala 20 25 30 Val Ile Gly Gly Ala Leu Gly Gly Leu GlyGly Ala Gly Gly Gly Cys 35 40 45 Ile Thr Gly Ala Ile Gly Ser Ile Trp AspGln Trp 50 55 60 32 161 DNA Brochocin-C immunity peptide; CDS (1)..(159)32 atg gta aaa act ata cta ttt tcg gtt gta att tca ttc gtt gca tta 48Met Val Lys Thr Ile Leu Phe Ser Val Val Ile Ser Phe Val Ala Leu 1 5 1015 tgt aac ttt tta ata aaa aaa gat gtg tct tca aaa aaa aaa tta ttt 96Cys Asn Phe Leu Ile Lys Lys Asp Val Ser Ser Lys Lys Lys Leu Phe 20 25 30tta aca ggt tct att gct gtc ttt cta att atc tat gat ttt cta tgg 144 LeuThr Gly Ser Ile Ala Val Phe Leu Ile Ile Tyr Asp Phe Leu Trp 35 40 45 attata ttc tct aac ta 161 Ile Ile Phe Ser Asn 50 33 53 PRT Brochocin-Cimmunity peptide; 33 Met Val Lys Thr Ile Leu Phe Ser Val Val Ile Ser PheVal Ala Leu 1 5 10 15 Cys Asn Phe Leu Ile Lys Lys Asp Val Ser Ser LysLys Lys Leu Phe 20 25 30 Leu Thr Gly Ser Ile Ala Val Phe Leu Ile Ile TyrAsp Phe Leu Trp 35 40 45 Ile Ile Phe Ser Asn 50 34 53 PRT Brochocin-Cimmunity peptide 34 Met Val Lys Thr Ile Leu Phe Ser Val Val Ile Ser PheVal Ala Leu 1 5 10 15 Cys Asn Phe Leu Ile Lys Lys Asp Val Ser Ser LysLys Lys Leu Phe 20 25 30 Leu Thr Gly Ser Ile Ala Val Phe Leu Ile Ile TyrAsp Phe Leu Trp 35 40 45 Ile Ile Phe Ser Asn 50 35 2226 DNA Enterocin900 operon; 35 aagctttact tgatattagt tctgagttct gcctgattta tcagtaacataactctagag 60 ataactgcgt cgctatctca agttcttttt tcttttctta caaaataaatatactttatt 120 tcattttata agtcaacgtt ttcattgctt ataaattagt ttttttaatcatctcagtaa 180 taatttgcta tgtcagttcg atcaatacca tttgcatgaa agtacagctataagccaatc 240 acacctaacc tactcttaat cgtataatga ttccagttag caaggtctttaatactcatg 300 atccccatct ggttgagttt ttttcattcg aaaccctatg ccccaaaagaccgtcatttt 360 aggaatattc catactttct caggaacatt ttgatacgtc cattcagcaataaacccttc 420 attgtgcttc gcttcattgt ctaaggcgag tttggccaaa ggggattatctccgcgacac 480 ctaccgtagc aatcaatcct aattcttctt taatacgttc cttggatcatttgaacgaat 540 tttttccttc tctgacttct tgttccttca gtcgtaaaaa tattcagtgatctggtcact 600 tttaaaatgg gttcatcgat tggatacatc agtagatctt cgtcagccacatatcttttg 660 aaaatattat ttacccgcat atttcgcttg atatataggt tcatacgtggtggaacaacg 720 tatgatgttt taggaaatag ttgtgataaa tcacgtggtc tactcacatttgtaatatca 780 taccgctttt ttgcttcagg agaagaagct ctaatatcaa tcctaaaccagtattgtcag 840 cgcgactcat aacaacaagt tctgttgtta atggatcaaa atttctttctatacactcga 900 tactcgcata aaaaggcttc atgtcgatta gaaaataatc atttactgattctttcgaat 960 aatccagcat gaataacacc cattcttttt cacattacac aaacgtaagttaggaaatat 1020 aaagaagaaa actaaatagc actaaacaaa caagacaact catgcttattccgtataaga 1080 aactacatat tatgttaact agttattaaa ataacatatt taataaaattaaattgtgat 1140 tttataggtt tcaggaatga aaaagcctta tttcaggaag tttttaactgtttgctatag 1200 atgtatgtca tgatagcatc gtaataaaaa tactctaaaa ggagcgagtttaaatatgca 1260 aaatgtaaaa gaattaagta cgaaagagat gaaacaaatt atcggtggagaaaatgatca 1320 cagaatgcct aatgagttaa atagacctaa caacttatct aaaggtggagcaaaatgtgg 1380 tgctgcaatt gctgggggat tatttggaat cccaaaagga ccactagcatgggctgctgg 1440 gttagcaaat gtatactcta aatgcaacta aaaaagaaga gaaaaaactcattacgagtt 1500 ttttctcttc tttttttgca tgaaattagg aataactaat aaaacaatagcaatcaatag 1560 taaaatctta cttaatatag tttcggaaaa aataaataat cctaaatttataattactgc 1620 taaaaaaatg cataaattat actctaaatt attttttttt aaattcataatataaacatc 1680 ctctctttaa ttagtctacc attccgaaat atttcatccc cagctctttttttactaata 1740 taccaactac atttaataac aaaataacta gtaaacttaa tatttttagtggcatagaat 1800 attcaaaaat aaataaaggc accatacatg tagctatcaa tataaatacagaacttacgt 1860 attttattat tttacggaac attataacct attacaactc cgcaaatagccatagcccat 1920 accatagata agatttttac cagcaccacc accacatgtt tgttttatctctttcatact 1980 taattttttt acattttgca tgtctctaca tgctcctttt aaagtttttttagaacctca 2040 cgactataac atggataatt taatcgtggt caaaaacttc ctgaaatagggtgtttcata 2100 tcctgaacac gaatttttag tcaattttcg aaaaatgaaa ctttaaaatttctttgacca 2160 gaactctatt tattcttgtg ttgttccttc gaataggttc ccgtatatcttttttatttg 2220 aagctt 2226 36 215 DNA Enterocin 900 peptide; CDS(1)..(213) 36 atg caa aat gta aaa gaa tta agt acg aaa gag atg aaa caaatt atc 48 Met Gln Asn Val Lys Glu Leu Ser Thr Lys Glu Met Lys Gln IleIle 1 5 10 15 ggt gga gaa aat gat cac aga atg cct aat gag tta aat agacct aac 96 Gly Gly Glu Asn Asp His Arg Met Pro Asn Glu Leu Asn Arg ProAsn 20 25 30 aac tta tct aaa ggt gga gca aaa tgt ggt gct gca att gct ggggga 144 Asn Leu Ser Lys Gly Gly Ala Lys Cys Gly Ala Ala Ile Ala Gly Gly35 40 45 tta ttt gga atc cca aaa gga cca cta gca tgg gct gct ggg tta gca192 Leu Phe Gly Ile Pro Lys Gly Pro Leu Ala Trp Ala Ala Gly Leu Ala 5055 60 aat gta tac tct aaa tgc aac ta 215 Asn Val Tyr Ser Lys Cys Asn 6570 37 71 PRT Enterocin 900 peptide; 37 Met Gln Asn Val Lys Glu Leu SerThr Lys Glu Met Lys Gln Ile Ile 1 5 10 15 Gly Gly Glu Asn Asp His ArgMet Pro Asn Glu Leu Asn Arg Pro Asn 20 25 30 Asn Leu Ser Lys Gly Gly AlaLys Cys Gly Ala Ala Ile Ala Gly Gly 35 40 45 Leu Phe Gly Ile Pro Lys GlyPro Leu Ala Trp Ala Ala Gly Leu Ala 50 55 60 Asn Val Tyr Ser Lys Cys Asn65 70 38 71 PRT Enterocin 900 peptide 38 Met Gln Asn Val Lys Glu Leu SerThr Lys Glu Met Lys Gln Ile Ile 1 5 10 15 Gly Gly Glu Asn Asp His ArgMet Pro Asn Glu Leu Asn Arg Pro Asn 20 25 30 Asn Leu Ser Lys Gly Gly AlaLys Cys Gly Ala Ala Ile Ala Gly Gly 35 40 45 Leu Phe Gly Ile Pro Lys GlyPro Leu Ala Trp Ala Ala Gly Leu Ala 50 55 60 Asn Val Tyr Ser Lys Cys Asn65 70 39 103 PRT Colicin V pre-peptide; DISULFID (91)..(102) 39 Met ArgThr Leu Thr Leu Asn Glu Leu Asp Ser Val Ser Gly Gly Ala 1 5 10 15 SerGly Arg Asp Ile Ala Met Ala Ile Gly Thr Leu Ser Gly Gln Phe 20 25 30 ValAla Gly Gly Ile Gly Ala Ala Ala Gly Gly Val Ala Gly Gly Ala 35 40 45 IleTyr Asp Tyr Ala Ser Thr His Lys Pro Asn Pro Ala Met Ser Pro 50 55 60 SerGly Leu Gly Gly Thr Ile Lys Gln Lys Pro Glu Gly Ile Pro Ser 65 70 75 80Glu Ala Trp Asn Tyr Ala Ala Gly Arg Leu Cys Asn Trp Ser Pro Asn 85 90 95Asn Leu Ser Asp Val Cys Leu 100 40 88 PRT Colicin V; DISULFID (76)..(87)40 Ala Ser Gly Arg Asp Ile Ala Met Ala Ile Gly Thr Leu Ser Gly Gln 1 510 15 Phe Val Ala Gly Gly Ile Gly Ala Ala Ala Gly Gly Val Ala Gly Gly 2025 30 Ala Ile Tyr Asp Tyr Ala Ser Thr His Lys Pro Asn Pro Ala Met Ser 3540 45 Pro Ser Gly Leu Gly Gly Thr Ile Lys Gln Lys Pro Glu Gly Ile Pro 5055 60 Ser Glu Ala Trp Asn Tyr Ala Ala Gly Arg Leu Cys Asn Trp Ser Pro 6570 75 80 Asn Asn Leu Ser Asp Val Cys Leu 85 41 675 DNA carnobacteriocinBM1; RBS (89)..(93) 41 tcgagatacg tttatccatg gttcaggatg attttatcaacgagttaatt atttatgcta 60 cagtaaactt gttactaaat actttataag gagtgtatgt acatg aaa agc gtt 114 Met Lys Ser Val 1 aaa gaa cta aat aaa aaa gaa atgcaa caa att aat ggt gga gct atc 162 Lys Glu Leu Asn Lys Lys Glu Met GlnGln Ile Asn Gly Gly Ala Ile 5 10 15 20 tct tat ggc aat ggt gtt tat tgtaac aaa gag aaa tgt tgg gta aac 210 Ser Tyr Gly Asn Gly Val Tyr Cys AsnLys Glu Lys Cys Trp Val Asn 25 30 35 aag gca gaa aac aaa caa gct att actgga ata gtt atc ggt gga tgg 258 Lys Ala Glu Asn Lys Gln Ala Ile Thr GlyIle Val Ile Gly Gly Trp 40 45 50 gct tct agt tta gca gga atg gga cattaaagaggta tctagtt atg ata 308 Ala Ser Ser Leu Ala Gly Met Gly His MetIle 55 60 aaa gat gaa aaa ata aat aaa atc tat gct tta gtt aag agc gcactt 356 Lys Asp Glu Lys Ile Asn Lys Ile Tyr Ala Leu Val Lys Ser Ala Leu65 70 75 gat aat acg gat gtt aag aat gat aaa aaa ctt tct tta ctt ctt atg404 Asp Asn Thr Asp Val Lys Asn Asp Lys Lys Leu Ser Leu Leu Leu Met 8085 90 95 aga ata caa gaa aca tca att aat gga gaa cta ttt tac gat tat aaa452 Arg Ile Gln Glu Thr Ser Ile Asn Gly Glu Leu Phe Tyr Asp Tyr Lys 100105 110 aaa gaa tta cag cca gct att agt atg tac tct att caa cat aac ttt500 Lys Glu Leu Gln Pro Ala Ile Ser Met Tyr Ser Ile Gln His Asn Phe 115120 125 cgg gtt cct gac gat cta gta aaa ctg tta gca tta gtt caa aca cct548 Arg Val Pro Asp Asp Leu Val Lys Leu Leu Ala Leu Val Gln Thr Pro 130135 140 aaa gct tgg tca ggg ttt taactttagt tccagatgag ttaaaatcct 596 LysAla Trp Ser Gly Phe 145 taaaaataag gaataatggt aaatcagcat tccttatttttatagtcatc acactataac 656 tttacttaaa gatgttcga 675 42 61 PRTcarnobacteriocin BM1; 42 Met Lys Ser Val Lys Glu Leu Asn Lys Lys Glu MetGln Gln Ile Asn 1 5 10 15 Gly Gly Ala Ile Ser Tyr Gly Asn Gly Val TyrCys Asn Lys Glu Lys 20 25 30 Cys Trp Val Asn Lys Ala Glu Asn Lys Gln AlaIle Thr Gly Ile Val 35 40 45 Ile Gly Gly Trp Ala Ser Ser Leu Ala Gly MetGly His 50 55 60 43 88 PRT carnobacteriocin BM1; 43 Met Ile Lys Asp GluLys Ile Asn Lys Ile Tyr Ala Leu Val Lys Ser 1 5 10 15 Ala Leu Asp AsnThr Asp Val Lys Asn Asp Lys Lys Leu Ser Leu Leu 20 25 30 Leu Met Arg IleGln Glu Thr Ser Ile Asn Gly Glu Leu Phe Tyr Asp 35 40 45 Tyr Lys Lys GluLeu Gln Pro Ala Ile Ser Met Tyr Ser Ile Gln His 50 55 60 Asn Phe Arg ValPro Asp Asp Leu Val Lys Leu Leu Ala Leu Val Gln 65 70 75 80 Thr Pro LysAla Trp Ser Gly Phe 85 44 61 PRT Carnobacterium B2 44 Met Lys Ser ValLys Glu Leu Asn Lys Lys Glu Met Gln Gln Ile Asn 1 5 10 15 Gly Gly AlaIle Ser Tyr Gly Asn Gly Val Tyr Cys Asn Lys Glu Lys 20 25 30 Cys Trp ValAsn Lys Ala Glu Asn Lys Gln Ala Ile Thr Gly Ile Val 35 40 45 Ile Gly GlyTrp Ala Ser Ser Leu Ala Gly Met Gly His 50 55 60 45 88 PRTCarnobacterium B2 45 Met Ile Lys Asp Glu Lys Ile Asn Lys Ile Tyr Ala LeuVal Lys Ser 1 5 10 15 Ala Leu Asp Asn Thr Asp Val Lys Asn Asp Lys LysLeu Ser Leu Leu 20 25 30 Leu Met Arg Ile Gln Glu Thr Ser Ile Asn Gly GluLeu Phe Tyr Asp 35 40 45 Tyr Lys Lys Glu Leu Gln Pro Ala Ile Ser Met TyrSer Ile Gln His 50 55 60 Asn Phe Arg Val Pro Asp Asp Leu Val Lys Leu LeuAla Leu Val Gln 65 70 75 80 Thr Pro Lys Ala Trp Ser Gly Phe 85 46 1907DNA carnobacterium B2 operon; -35_signal (165)..(170) 46 aagcttttatagtacaatta tttatgcgtg ctatgcaata gctattgtat atactatttt 60 tactatgagaaaagattctt atgaaaataa caaaaataat cgtaaaaaag ttatatagca 120 tttatttcatttatgaattc aaataccctg gttcaagatg tattttccaa aaaaatgttc 180 agatatgatatagttttttt gaaatacaaa tataaaataa aggagtttga tttag atg 238 Met 1 aat agcgta aaa gaa tta aac gtg aaa gaa atg aaa caa tta cac ggt 286 Asn Ser ValLys Glu Leu Asn Val Lys Glu Met Lys Gln Leu His Gly 5 10 15 gga gta aattat ggt aat ggt gtt tct tgc agt aaa aca aaa tgt tca 334 Gly Val Asn TyrGly Asn Gly Val Ser Cys Ser Lys Thr Lys Cys Ser 20 25 30 gtt aac tgg ggacaa gcc ttt caa gaa aga tac aca gct gga att aac 382 Val Asn Trp Gly GlnAla Phe Gln Glu Arg Tyr Thr Ala Gly Ile Asn 35 40 45 tca ttt gta agt ggagtc gct tct ggg gca gga tcc att ggt agg aga 430 Ser Phe Val Ser Gly ValAla Ser Gly Ala Gly Ser Ile Gly Arg Arg 50 55 60 65 ccg taaatatataaatacaatat agagcaaggt ggtgataca atg gat ata aag 484 Pro Met Asp Ile Lys70 tct caa aca tta tat ttg aat cta agc gag gca tat aaa gac cct gaa 532Ser Gln Thr Leu Tyr Leu Asn Leu Ser Glu Ala Tyr Lys Asp Pro Glu 75 80 85gta aaa gct aat gaa ttc tta tca aaa tta gtt gta caa tgt gct ggg 580 ValLys Ala Asn Glu Phe Leu Ser Lys Leu Val Val Gln Cys Ala Gly 90 95 100aaa tta aca gct tca aac agt gag aac agt tat att gaa gta ata tca 628 LysLeu Thr Ala Ser Asn Ser Glu Asn Ser Tyr Ile Glu Val Ile Ser 105 110 115ttg cta tct agg ggt att tct agt tat tat tta tcc cat aaa cgt ata 676 LeuLeu Ser Arg Gly Ile Ser Ser Tyr Tyr Leu Ser His Lys Arg Ile 120 125 130att cct tca agt atg tta act ata tat act caa ata caa aag gat ata 724 IlePro Ser Ser Met Leu Thr Ile Tyr Thr Gln Ile Gln Lys Asp Ile 135 140 145150 aaa aac ggg aat att gac acc gaa aaa tta agg aaa tat gag ata gca 772Lys Asn Gly Asn Ile Asp Thr Glu Lys Leu Arg Lys Tyr Glu Ile Ala 155 160165 aaa gga tta atg tcc gtt cct tat ata tat ttc taattttttc aatgatgtta825 Lys Gly Leu Met Ser Val Pro Tyr Ile Tyr Phe 170 175 gttgacttcaaaaagatgtg aaatcgatta gcattttcaa aattagatta aaaatactat 885 ctatataaaatagaactact gatttaaagt atttataaga atataaagta gcaaataaca 945 tgatagacacaattaaggag cgacatttta tggaaaattt gaaatggtat tcgggcggga 1005 acgatagaaaaaaaaagca atg gct act att act gat ttg tta aac gat tta 1057 Met Ala ThrIle Thr Asp Leu Leu Asn Asp Leu 180 185 aaa ata gac tta ggt aac gaa tctcta caa aat gtc tta gaa aat tat 1105 Lys Ile Asp Leu Gly Asn Glu Ser LeuGln Asn Val Leu Glu Asn Tyr 190 195 200 ctt gaa gaa ttg gaa caa gca aatgct gct gtt cca att ata tta ggc 1153 Leu Glu Glu Leu Glu Gln Ala Asn AlaAla Val Pro Ile Ile Leu Gly 205 210 215 220 cgt atg aac ata gat atc tctaca gca atc aga aaa gat ggt gtt act 1201 Arg Met Asn Ile Asp Ile Ser ThrAla Ile Arg Lys Asp Gly Val Thr 225 230 235 tta tca gaa att cag tct aaaaaa tta aaa gag ctg att tca ata tcc 1249 Leu Ser Glu Ile Gln Ser Lys LysLeu Lys Glu Leu Ile Ser Ile Ser 240 245 250 tat att aaa tat ggc tattaatttagta ttaataacag tgtaggattg 1297 Tyr Ile Lys Tyr Gly Tyr 255attcaaatta tttgaatcaa aatttatata caaattttat ttattttggg tctttaaata 1357attttgtgta agttcaaatt atttaaagat gagttaaaac tctatcttcg aaaaacatca 1417caaaatgtga tgaaatttgt ccccaatttt ggaccttcat ggtccatttt ttcgttacat 1477ccatcgtcac taaacaaagc atttttagta aggattcatc agatgggaat actaccttag 1537attttgttgg ctttcacagc tgacaatgga ggccttcaat cacattggcg gtataaataa 1597tccggcgcaa atctgctgaa tacttgaaaa atgtcgctaa ttctgcccag ttatctatca 1657attcttcaat taatttgtct gcgtttatat ccattttcat tccccttttt taatttttca 1717ttttttagtt actttaaacg gtttaaagcc ttaagcactt aggctttaat cttttttcac 1777ttgatctaat tatttgaact tcagcattta tcttttgatt tattctttta gggaattgac 1837cgaataggga gatttcctgt gagtaggcgc caacggtggt ggcggtcgga gtcagccgac 1897tcacaagctt 1907 47 66 PRT carnobacterium B2 operon; 47 Met Asn Ser ValLys Glu Leu Asn Val Lys Glu Met Lys Gln Leu His 1 5 10 15 Gly Gly ValAsn Tyr Gly Asn Gly Val Ser Cys Ser Lys Thr Lys Cys 20 25 30 Ser Val AsnTrp Gly Gln Ala Phe Gln Glu Arg Tyr Thr Ala Gly Ile 35 40 45 Asn Ser PheVal Ser Gly Val Ala Ser Gly Ala Gly Ser Ile Gly Arg 50 55 60 Arg Pro 6548 111 PRT carnobacterium B2 operon; 48 Met Asp Ile Lys Ser Gln Thr LeuTyr Leu Asn Leu Ser Glu Ala Tyr 1 5 10 15 Lys Asp Pro Glu Val Lys AlaAsn Glu Phe Leu Ser Lys Leu Val Val 20 25 30 Gln Cys Ala Gly Lys Leu ThrAla Ser Asn Ser Glu Asn Ser Tyr Ile 35 40 45 Glu Val Ile Ser Leu Leu SerArg Gly Ile Ser Ser Tyr Tyr Leu Ser 50 55 60 His Lys Arg Ile Ile Pro SerSer Met Leu Thr Ile Tyr Thr Gln Ile 65 70 75 80 Gln Lys Asp Ile Lys AsnGly Asn Ile Asp Thr Glu Lys Leu Arg Lys 85 90 95 Tyr Glu Ile Ala Lys GlyLeu Met Ser Val Pro Tyr Ile Tyr Phe 100 105 110 49 81 PRT carnobacteriumB2 operon; 49 Met Ala Thr Ile Thr Asp Leu Leu Asn Asp Leu Lys Ile AspLeu Gly 1 5 10 15 Asn Glu Ser Leu Gln Asn Val Leu Glu Asn Tyr Leu GluGlu Leu Glu 20 25 30 Gln Ala Asn Ala Ala Val Pro Ile Ile Leu Gly Arg MetAsn Ile Asp 35 40 45 Ile Ser Thr Ala Ile Arg Lys Asp Gly Val Thr Leu SerGlu Ile Gln 50 55 60 Ser Lys Lys Leu Lys Glu Leu Ile Ser Ile Ser Tyr IleLys Tyr Gly 65 70 75 80 Tyr 50 66 PRT Carnobacterium B2 operon 50 MetAsn Ser Val Lys Glu Leu Asn Val Lys Glu Met Lys Gln Leu His 1 5 10 15Gly Gly Val Asn Tyr Gly Asn Gly Val Ser Cys Ser Lys Thr Lys Cys 20 25 30Ser Val Asn Trp Gly Gln Ala Phe Gln Glu Arg Tyr Thr Ala Gly Ile 35 40 45Asn Ser Phe Val Ser Gly Val Ala Ser Gly Ala Gly Ser Ile Gly Arg 50 55 60Arg Pro 65 51 111 PRT Carnobacterium B2 operon 51 Met Asp Ile Lys SerGln Thr Leu Tyr Leu Asn Leu Ser Glu Ala Tyr 1 5 10 15 Lys Asp Pro GluVal Lys Ala Asn Glu Phe Leu Ser Lys Leu Val Val 20 25 30 Gln Cys Ala GlyLys Leu Thr Ala Ser Asn Ser Glu Asn Ser Tyr Ile 35 40 45 Glu Val Ile SerLeu Leu Ser Arg Gly Ile Ser Ser Tyr Tyr Leu Ser 50 55 60 His Lys Arg IleIle Pro Ser Ser Met Leu Thr Ile Tyr Thr Gln Ile 65 70 75 80 Gln Lys AspIle Lys Asn Gly Asn Ile Asp Thr Glu Lys Leu Arg Lys 85 90 95 Tyr Glu IleAla Lys Gly Leu Met Ser Val Pro Tyr Ile Tyr Phe 100 105 110 52 81 PRTCarnobacterium B2 operon 52 Met Ala Thr Ile Thr Asp Leu Leu Asn Asp LeuLys Ile Asp Leu Gly 1 5 10 15 Asn Glu Ser Leu Gln Asn Val Leu Glu AsnTyr Leu Glu Glu Leu Glu 20 25 30 Gln Ala Asn Ala Ala Val Pro Ile Ile LeuGly Arg Met Asn Ile Asp 35 40 45 Ile Ser Thr Ala Ile Arg Lys Asp Gly ValThr Leu Ser Glu Ile Gln 50 55 60 Ser Lys Lys Leu Lys Glu Leu Ile Ser IleSer Tyr Ile Lys Tyr Gly 65 70 75 80 Tyr 53 35 DNA JMc7; 53 cccaagcttctgctgtaaat tatggtaatg gtgtt 35 54 36 DNA KLR179; 54 gcgcaagcttctgctcggac accagaaatg cctgtt 36 55 30 DNA KLR180; 55 ggccaagcttgccattaagt ctggttgcta 30 56 20 DNA MB32; 56 aattcgagct cgcccaaatc 20 5740 DNA MB37; 57 tgagtaattt tcggtgcagc acctcctacg acttgttcga 40 58 23 DNARW58; 58 tacgcgcaag aacagacaaa atc 23 59 40 DNA MB38; 59 tgagtaattttcggtgcagc tcctccgtta gcttctgaaa 40 60 19 DNA MB39; 60 tacgaattcgagctcgccc 19 61 76 DNA MB42; 61 attttcggtg cagcacctcc agaaacagaatctaattcat ttagagtcag agttctcata 60 ataactttcc tctttt 76 62 40 DNA MB41;62 tgagtaattt tcggtgcagc cataataact ttcctctttt 40 63 37 DNA MB43; 63atatcacgcc ctgaagcacc tcctacgact tgttcga 37 64 36 DNA MB44;misc_difference (11)..(12) standard_name= +37any base” 64 aattaagcttggatccttct gtgtggattg tccaat 36 65 21 DNA APO-1 misc_difference(11)..(12) standard_name=“(any base)” 65 aaagatattg gaaaggattg g 21 66 7PRT Carnobacterium sp. 66 Val Asn Tyr Gly Asn Gly Val 1 5 67 32 PRT CF01probe MISC_FEATURE (13)..(13) C or A 67 Gly Ala Ala Ala Ala Thr Gly AlaThr Cys Ala Thr Xaa Gly Xaa Ala 1 5 10 15 Thr Gly Cys Cys Xaa Ala AlaThr Gly Ala Ala Cys Thr Xaa Ala Ala 20 25 30 68 61 PRT Leucocin A 68 MetMet Asn Met Lys Pro Thr Glu Ser Tyr Glu Gln Leu Asp Asn Ser 1 5 10 15Ala Leu Glu Gln Val Val Gly Gly Lys Tyr Tyr Gly Asn Gly Val His 20 25 30Cys Thr Lys Ser Gly Cys Ser Val Asn Trp Gly Glu Ala Glu Ser Ala 35 40 45Gly Val His Arg Leu Ala Asn Gly Gly Asn Gly Phe Trp 50 55 60 69 36 PRTMesenteriocin Y105 69 Lys Tyr Tyr Gly Asn Gly Val His Cys Thr Lys SerGly Cys Ser Val 1 5 10 15 Asn Trp Gly Glu Ala Ala Ser Ala Gly Ile HisArg Leu Ala Asn Gly 20 25 30 Gly Asn Gly Phe 35 70 41 PRT Sakacin PMISC_FEATURE (33)..(33) any amino acid 70 Lys Tyr Tyr Gly Asn Gly ValHis Cys Gly Lys His Ser Cys Thr Val 1 5 10 15 Asp Trp Gly Thr Ala IleGly Asn Ile Gly Asn Asn Ala Ala Ala Asn 20 25 30 Xaa Ala Thr Gly Xaa AsnAla Xaa Xaa 35 40 71 62 PRT Pediocin PA1 71 Met Lys Lys Ile Glu Lys LeuThr Glu Lys Glu Met Ala Asn Ile Ile 1 5 10 15 Gly Gly Lys Tyr Tyr GlyAsn Gly Val Thr Cys Gly Lys His Ser Cys 20 25 30 Ser Val Asp Trp Gly LysAla Thr Thr Cys Ile Ile Asn Asn Gly Ala 35 40 45 Met Ala Trp Ala Thr GlyGly His Gln Gly Asn His Lys Cys 50 55 60 72 66 PRT Carnobacteriocin B272 Met Asn Ser Val Lys Glu Leu Asn Val Lys Glu Met Lys Gln Leu His 1 510 15 Gly Gly Val Asn Tyr Gly Asn Gly Val Ser Cys Ser Lys Thr Lys Cys 2025 30 Ser Val Asn Trp Gly Gln Ala Phe Gln Glu Arg Tyr Thr Ala Gly Ile 3540 45 Asn Ser Phe Val Ser Gly Val Ala Ser Gly Ala Gly Ser Ile Gly Arg 5055 60 Arg Pro 65 73 61 PRT Carnobacteriocin BM1 73 Met Lys Ser Val LysGlu Leu Asn Lys Lys Glu Met Gln Gln Ile Ile 1 5 10 15 Gly Gly Ala IleSer Tyr Gly Asn Gly Val Tyr Cys Asn Lys Glu Lys 20 25 30 Cys Trp Val AsnLys Ala Glu Asn Lys Gln Ala Ile Thr Gly Ile Val 35 40 45 Ile Gly Gly TrpAla Ser Ser Leu Ala Gly Met Gly His 50 55 60 74 59 PRT Sakacin A 74 MetAsn Asn Val Lys Glu Leu Ser Met Thr Glu Leu Gln Thr Ile Thr 1 5 10 15Gly Gly Ala Arg Ser Tyr Gly Asn Gly Val Tyr Cys Asn Asn Lys Lys 20 25 30Cys Trp Val Asn Arg Gly Glu Ala Thr Gln Ser Ile Ile Gly Gly Met 35 40 45Ile Ser Gly Trp Ala Ser Gly Leu Ala Gly Met 50 55 75 31 PRT Curvacin AMISC_FEATURE (25)..(25) any amino acid 75 Ala Cys Ser Tyr Gly Asn GlyVal Tyr Cys Asn Asn Lys Lys Ser Trp 1 5 10 15 Val Asn Gly Gly Glu AlaThr Gln Xaa Xaa Ile Xaa Xaa Gly Xaa 20 25 30 76 18 PRT CarnobacteriocinA 76 Met Asn Asn Val Lys Glu Leu Ser Ile Lys Glu Met Gln Gln Val Thr 1 510 15 Gly Gly 77 18 PRT Lactacin F 77 Met Lys Gln Phe Asn Tyr Leu SerHis Lys Asp Leu Ala Val Val Val 1 5 10 15 Gly Gly 78 21 PRT LactococcinB 78 Met Lys Asn Gln Leu Asn Phe Asn Ile Leu Ser Glu Glu Asp Leu Ala 1 510 15 Glu Ala Asn Gly Gly 20 79 21 PRT Lactococcin A 79 Met Lys Asn GlnLeu Asn Phe Asn Ile Leu Ser Glu Glu Asp Leu Ser 1 5 10 15 Glu Ala AsnGly Gly 20 80 21 PRT Lactococcin M 80 Met Lys Asn Gln Leu Asn Phe GluIle Leu Ser Glu Glu Asp Leu Gln 1 5 10 15 Gly Ile Asn Gly Gly 20

What is claimed:
 1. An isolated peptide inhibitory to bacterial growthselected from the group consisting of enterocin 900 (SEQ ID NO:28),brochocin-C (SEQ ID NO:23), and muteins thereof.
 2. The peptide of claim1, wherein said peptide is enterocin 900 or a mutein thereof.
 3. Thepeptide of claim 1, wherein said peptide is brochocin-C peptide A,brochocin-C peptide B, or a mutein thereof.
 4. An isolatedpolynucleotide, said polynucleotide comprising a structural genesequence which encodes a peptide inhibitory to bacterial growth selectedfrom the group consisting of enterocin 900 (SEQ ID NO:28), brochocin-C(SEQ ID NO:23), and muteins thereof.
 5. The polynucleotide of claim 4,further comprising a promoter operable in a host microorganism, operablylinked to said structural gene sequence.
 6. The polynucleotide of claim5, wherein said microorganism comprises a lactic acid bacterium.
 7. Thepolynucleotide of claim 6, further comprising a bacteriocin processingpeptide operable in said host microorganism, operably linked to saidstructural gene sequence.
 8. The polynucleotide of claim 10, whereinsaid bacteriocin processing peptide comprises a divergicin A processingpeptide.
 9. A secretion vector, for providing secretion of aheterologous protein from a microbial host cell, said secretion vectorcomprising: a first polynucleotide encoding a heterologous protein; asecond polynucleotide encoding a bacteriocin processing peptide operablein said host cell, operably linked to said polynucleotide encoding saidheterologous protein; and a promoter operable in said host cell,operably linked to said polynucleotide encoding said heterologousprotein.
 10. The secretion vector of claim 9, wherein said bacteriocinprocessing peptide comprises a divergicin A processing peptide or anoperable mutein thereof.
 11. The secretion vector of claim 9, whereinsaid first polynucleotide encodes a heterologous bacteriocin.
 12. Thesecretion vector of claim 11, further comprising an immunity gene whichconfers immunity from said heterologous bacteriocin to said host cell.13. The secretion vector of claim 11, wherein said vector encodes aplurality of different bacteriocins.
 14. The vector pCD3.4 (SEQ IDNO:14).
 15. A host cell transformed with the secretion vector of claim12.
 16. A host cell transformed with a plurality of secretion vectors ofclaim
 12. 17. A host cell transformed with the secretion vector of claim13.
 18. A method for inhibiting the growth of susceptible bacteria in anenvironment, comprising: providing a microorganism capable of expressinga bacteriocin selected from the group consisting of enterocin 900,brochocin-C, and operable muteins thereof; and applying saidmicroorganism to said environment in an amount sufficient to inhibit thegrowth of susceptible bacteria.
 19. The method of claim 18, wherein saidenvironment comprises a foodstuff.
 20. The method of claim 19, whereinsaid foodstuff comprises meat.
 21. The method of claim 18, wherein saidenvironment comprises a living mammal.
 22. The method of claim 21,wherein said environment comprises a food preparation area.
 23. Themethod of claim 21, wherein said microorganism expresses a plurality ofbacteriocins.
 24. The method of claim 21, wherein said environmentcomprises a fermentation vessel.
 25. A method for inhibiting the growthof susceptible bacteria in an environment, comprising: providing abactericin selected from the group consisting of enterocin 900,brochocin-C, and operable muteins thereof; and applying said bacteriocinto said environment in an amount sufficient to inhibit the growth ofsusceptible bacteria.
 26. The method of claim 25, wherein saidbacteriocin inhibits bacteria which cause mastitis.
 27. A method forobtaining secretion of a protein from a microorganism, said methodcomprising: providing a secretion vector comprising a promoter operablein said microorganism, operably linked to a polynucleotide encoding abacteriocin processing peptide operable in said microorganism, operablylinked to a polynucleotide encoding said protein; transforming saidmicroorganism with said secretion vector; and culturing said transformedmicroorganism under conditions that induce expression and secretion. 28.The method of claim 27, wherein said bacteriocin processing peptidecomprises a divergicin A processing peptide.
 30. An expression vectorfor secreting a heterologous protein in a microorganism host, saidvector comprising: a polynucleotide encoding a promoter operable in saidhost; a polynucleotide encoding a leucocin A processing peptide; apolynucleotide encoding said heterologous protein; a polynucleotideencoding lcac (SEQ ID NO:4); and a polynucleotide encoding lcaD (SEQ IDNO:5).
 31. The vector of claim 30, wherein said heterologous proteincomprises leucocin A.
 32. An antibody specific for a bacteriocinselected from the group consisting of brochocin-C and enterocin
 900. 33.A method for detecting expression of a bacteriocin in a host cell, saidmethod comprising: providing an antibody specific for a bacteriocinselected from the group consisting of brochocin-C and enterocin 900;contacting said host cell with said antibody; and detecting binding orabsence of binding between said antibody and said bacteriocin.
 34. Anisolated polynucleotide, said polynucleotide comprising a sequenceencoding brochocin-C immunity (SEQ ID NO:26) or an operable muteinthereof.
 35. A method for purifying brochocin-C from a microorganismculture, comprising: extracting said microorganism culture withn-butanol; removing said n-butanol to provide an extract; and filteringsaid extract by gel filtration.
 36. A method for purifying enterocin 900from a solution, comprising: applying said solution to a hydrophobicinteraction column; washing said column with a solution having less than40% ethanol; eluting partially purified enterocin 900 from said columnwith a solution having about 40% or more ethanol; applying saidpartially purified enterocin 900 to an ion exchange column; washing saidcolumn with a salt solution having a concentration less than about 150mM; eluting enterocin 900 using a salt solution having a concentrationof about 200 mM.
 37. The method of claim 36, further comprising:applying enterocin 900 to a reverse-phase HPLC column; washing saidcolumn with a solution comprising less than about 50% ethanol; andeluting enterocin 900 using a solution comprising about 70% ethanol. 38.A method for inhibiting the growth of susceptible bacteria in anenvironment, comprising: providing a microorganism comprising asecretion vector, said secretion vector comprising: a firstpolynucleotide encoding a bacteriocin; a second polynucleotide encodinga bacteriocin processing peptide operable in said host cell, operablylinked to said polynucleotide encoding said bacteriocin; and a promoteroperable in said host cell, operably linked to said polynucleotideencoding said bacteriocin; and applying said microorganism to saidenvironment in an amount sufficient to inhibit the growth of susceptiblebacteria.
 39. The method of claim 38, wherein said bacteriocinprocessing peptide comprises a divergicin A processing peptide or anoperable mutein thereof.
 40. The method of claim 39, wherein saidsecretion vector further comprises an immunity gene which confersimmunity from said heterologous bacteriocin to said host cell.
 41. Themethod of claim 40, wherein said vector encodes a plurality of differentbacteriocins.